Klebsiella antigens

ABSTRACT

The present invention relates to isolated nucleic acid molecules which encode an antigen, a vector which comprises such nucleic acid molecule, and a host cell comprising such vector. Furthermore, the invention provides antigens from a  Klebsiella  species, as well as fragments and variants thereof, a process for producing such antigens, and a process for producing a cell, which expresses such antigen. Moreover, the present invention provides antibodies binding to such antigen, a hybridoma cell producing such antibodies, methods for producing such antibodies, a pharmaceutical composition comprising such nucleic acid molecule, antigen, vector or antibody, the use of such nucleic acid molecule, antigen, vector or antibody for the preparation of a pharmaceutical composition, methods for identifying an antagonist capable of binding such antigen or of reducing or inhibiting the interaction activity of such antigen, methods for diagnosing an infection and methods for the treatment or prevention of an infection. More specifically such antigens are produced by or associated with bacterial pathogens causing nosocomial infections or bacterial infections caused by  Klebsiella pneumoniae.

The present invention relates to isolated nucleic acid molecules whichencode an antigen, a vector which comprises such nucleic acid molecule,and a host cell comprising such vector. Furthermore, the inventionprovides antigens from a Klebsiella species, as well as fragments andvariants thereof, a process for producing such antigens, and a processfor producing a cell, which expresses such antigen. Moreover, thepresent invention provides antibodies binding to such antigen, ahybridoma cell producing such antibodies, methods for producing suchantibodies, a pharmaceutical composition comprising such nucleic acidmolecule, antigen, vector or antibody, the use of such nucleic acidmolecule, antigen, vector or antibody for the preparation of apharmaceutical composition, methods for identifying an antagonistcapable of binding such antigen or of reducing or inhibiting theinteraction activity of such antigen, methods for diagnosing aninfection and methods for the treatment or prevention of an infection.More specifically such antigens are produced by or associated withbacterial pathogens causing nosocomial infections or bacterialinfections caused by Klebsiella pneumoniae.

Klebsiella pneumoniae (K. pneumoniae) is a gram negative, facultativeanaerobic bacterium. Strains of K. pneumoniae are distinguished by thepresence of a capsular polysaccharide, of which there are 77 antigenictypes. This capsule encases the entire cell surface, accounts for thelarge appearance of the organism on gram stain, and provides resistanceagainst many host defense mechanisms. Colonies are large and highlymucoid. Klebsiella pneumoniae has the ability to fix nitrogen i.e. toconvert atmospheric nitrogen gas to ammonium.

Klebsiellae have two common habitats, one being the environment, wherethey are found in surface water, sewage and soil, and the other beingthe mucosal surface of mammals such as humans, horses or swine, whichthey colonize. In human, K. pneumoniae is present as a saprophyte in therespiratory, intestinal and urogenital tracts. When Klebsiella bacteriaget outside of the gut, however, serious infection can occur.

Klebsiella pneumoniae is a common hospital-acquired pathogen, causingurinary tract infections, nosocomial pneumonia, intraabdominalinfections, surgical wound infections and infection of the blood. All ofthese infections can progress to shock and death if not treated early inan aggressive fashion. K. pneumoniae is also a potentialcommunity-acquired pathogen. It is estimated that Klebsiella spp.account for 8% of endemic hospital infection and 3% of epidemicoutbreaks (Stamm E. et al., 1981).

Klebsiella's pathogenicity can be attributed to its production of aheat-stable enterotoxin. The virulence factors of K. pneumoniaeidentified so far include capsular polysaccharides (CPS),lipopolysaccharides, adhesins (type 1 and 3 pili, KPF-28 fimbria, CF29Kand aggregative adhesin) and iron acquisition systems (Podschun R etal., 1998).

K. pneumoniae infections are common in hospitals where they causepneumonia (characterized by emission of bloody sputum) and urinary tractinfections in catheterized patients. In fact, K. pneumoniae is secondonly to E. coli as a urinary tract pathogen. It accounts for 6 to 17percent of all nosocomial urinary tract infection (UTI). Klebsiellainfections are encountered far more often now than in the past. This isprobably due to the bacterium's antibiotic resistance properties.Klebsiella species may contain resistance plasmids (R-plasmids) whichconfer resistance to such antibiotics as Ampicillin and Carbenicillin(Wu et al., 2005). To make matters worse, the R-plasmids can betransferred to other enteric bacteria not necessarily of the samespecies. Hospital outbreaks of multidrug-resistant Klebsiella spp. areoften caused by a new type of strain, an ESBL producer (extendedspectrum β-lactamase). The incidence of ESBL-producing strains amongclinical Klebsiella isolates has been steadily increasing over the pastseveral years. Frequencies of up to 40% have been reported in certainregions. To treat K. pneumoniae infections, there are few antibioticsavailable like Cefepime, Polymyxin B (Parchuri et al., 2005), Carbapenem(Meropenem and Imipenem) (Ueda Y. et al., 2005).

There are attempts to develop a vaccice against Klebsiella. Among thedifferent bacterial constituents, two surface components are mainlybeing discussed as candidates for an anti-Klebsiella vaccine: LPS andCPS (Yadav et al., 2005). A great drawback of active immunization withLPS-containing vaccines is the induction of adverse toxic reactions,which are caused by the endotoxin content. CPS has been proven to behighly immunogenic and nontoxic. However, the serious disadvantage of aKlebsiella CPS vaccine is the great number of different K antigens (77as of today). There is a 6-valent Klebsiella CPS vaccine thatsubsequently was proven to be safe and immunogenic (Cryz et al., 1986),but covers only 30% of Klebsiella blood isolates, while a 25-valentvaccine would cover not more than 75%. Moreover, the production of suchmultivalent vaccines is difficult and expensive. To overcome thiscircumstance, protein based vaccines against Klebsiella have to bedeveloped. Kurupati et al. (2006) have identified a number ofimmunogenic antigens from Klebsiella pneumoniae, and two of thecandidate genes, namely OmpA and FepA, have been further characterizedin an in vivo mouse model. However, there are currently no prophylacticKlebsiella vaccines on the market or, according to public information,in active preclinical or clinical development. The Klebsiella vaccinedevelopment program (Klebgen Berna®) at Berna Biotech has beendiscontinued.

A vaccine can contain a whole variety of different antigens. Examples ofantigens are whole-killed or attenuated organisms, subfractions of theseorganisms/tissues, proteins, or, in their most simple form, peptides.Antigens can also be recognized by the immune system in form ofglycosylated proteins or peptides and may also be or containpolysaccharides or lipids. Short peptides can be used since for examplecytotoxic T-cells (CTL) recognize antigens in form of short, usually8-11 amino acids long peptides in conjunction with majorhistocompatibility complex (MHC). B-cells can recognize linear epitopesas short as 4-5 amino acids, as well as three-dimensional structures(conformational epitopes). In order to obtain sustained,antigen-specific immune responses, adjuvants need to trigger immunecascades that involve all cells of the immune system. Primarily,adjuvants are acting, but are not restricted in their mode of action, onso-called antigen presenting cells (APCs). These cells usually firstencounter the antigen(s) followed by presentation of processed orunmodified antigen to immune effector cells. Intermediate cell types mayalso be involved. Only effector cells with the appropriate specificityare activated in a productive immune response. The adjuvant may alsolocally retain antigens and co-injected other factors. In addition theadjuvant may act as a chemoattractant for other immune cells or may actlocally and/or systemically as a stimulating agent for the immunesystem.

There have been concerns to develop an inactivated whole cell vaccinefor humans because of the potential risk, that it may inducecross-reactive antibodies to human antigens. Therefore, subunit vaccinesare considered to have the greatest potential in preventing infectionsby Klebsiella pneumoniae.

The problem underlying the present invention was to provide means forthe development of pharmaceutical compositions such as vaccines againstnosocomial infections caused by Klebsiella. More particularly, theproblem was to provide an efficient, relevant and comprehensive set ofnucleic acid molecules or antigens, or fragments or variants thereof,from Klebsiella that can be used for the preparation of saidpharmaceutical compositions. A still further problem was to providemethods and means for producing an antigen, a fragment or variantthereof. Yet another problem was to provide pharmaceutical compositionscomprising said nucleic acids or said antigens. A still further problemof the invention was to provide antibodies, pharmaceutical compositionscomprising said antibodies, methods for the production of saidantibodies and the use of said antibodies for the preparation of apharmaceutical preparation. Furthermore, the object of the presentinvention was to provide methods for identifying an antagonist capableof binding an antigen, or a fragment or variant thereof, as well as toprovide methods for identifying an antagonist capable of reducing orinhibiting the interaction activity of such an antigen to itsinteraction partner. A further problem of the present invention was toprovide methods for diagnosing an infection with a Klebsiella organism.Still another problem underlying the invention was to provide methodsfor treating Klebsiella infections, and to provide methods forimmunizing an animal or human.

The problem underlying the present invention is solved in one aspect byan isolated nucleic acid molecule encoding an antigen or a fragmentthereof, comprising a nucleic acid sequence, which is selected from thegroup consisting of:

-   -   a) a nucleic acid molecule having at least 70% sequence identity        to a nucleic acid molecule having a nucleotide sequence selected        from the group comprising Seq ID Nos 1 to 187 and Seq ID No 375,    -   b) a nucleic acid molecule which is complementary to the nucleic        acid molecule of a),    -   c) a nucleic acid molecule comprising at least 15 sequential        bases of the nucleic acid molecule of a) or b),    -   d) a nucleic acid molecule which anneals under stringent        hybridisation conditions to the nucleic acid molecule of a), b),        or c),    -   e) a nucleic acid molecule which, but for the degeneracy of the        genetic code, would hybridise to the nucleic acid molecule        defined in a), b), c), or d).

In an embodiment of the invention the sequence identity to Seq ID Nos 1to 187 or Seq ID No 375 is at least 80%, more preferably at least 90%,still more preferably at least 95%, 96%, 97%, 98%, or 99%, or mostpreferably 100%.

In another embodiment the nucleic acid is DNA.

In an alternative embodiment the nucleic acid is RNA.

In still another embodiment the nucleic acid molecule is isolated from agenomic DNA, preferably from a species selected from the groupcomprising Klebsiella, preferably K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca. The nomenclature or classification ofKlebsiellae is used herein according to Ørskov, I. (1984).

In an embodiment of the invention the fragment is an active fragment oran active variant thereof.

In an embodiment the nucleic acid encodes an antigen or fragmentthereof, which comprises or consists of a polypeptide or peptidefragment from Klebsiella, preferably K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca.

The problem underlying the present invention is further solved by avector comprising a nucleic acid molecule as described above.

In an embodiment the vector is adapted for recombinant expression of theantigen, or fragment thereof, encoded by the nucleic acid molecule asdefined above.

The present invention also relates to a host cell comprising the vectoras defined above.

The problem underlying the present invention is solved in a furtheraspect by an antigen that is immunologically reactive with sera from ahuman having a Klebsiella infection, or an uninfected healthy human whowas previously infected with Klebsiella, wherein the antigen comprisesan isolated polypeptide or an active fragment or an active variantthereof from Klebsiella, preferably K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca.

The term “uninfected healthy human” as used herein comprises thoseindividuals who have or had multiple encounters with the pathogen, whichmay result in colonization, but which either do not result in anysymptoms, or which result in mild diseases. Said term and the rationaleof selecting sera of uninfected healthy humans for antigenidentification is further defined in Nagy, E. et al. (2003).

Another aspect of the present invention relates to an antigen,comprising or consisting of an isolated polypeptide selected from thegroup consisting of Seq ID Nos 188 to 374 and Seq ID No 376, or anactive fragment or an active variant thereof.

In an embodiment of the invention said polypeptide is encoded by anucleic acid molecule as defined above.

In another embodiment the active fragment of the antigen consists of atleast 50%, especially at least 60%, preferably at least 70%, morepreferably at least 80%, still more preferably at least 90%, even morepreferably at least 95%, 96%, 97% or 98%, most preferably 99% of saidpolypeptide, especially of a polypeptide as defined by any of the Seq IDNos 188 to 374 or Seq ID No 376.

In another embodiment the active variant of the antigen has at least50%, especially at least 60%, preferably at least 70%, more preferablyat least 80%, still more preferably at least 90%, even more preferablyat least 95%, 96%, 97% or 98%, most preferably 99% sequence identity tothe polypeptide, especially to a polypeptide as defined by any of theSeq ID Nos 188 to 374 or Seq ID No 376.

In one embodiment of the present invention the active fragment of theantigen comprises or consists of amino acids 2-130 of Seq ID No 205;amino acids 26-356 of Seq ID No 216; amino acids 2-180 of Seq ID No 223;amino acids 1-168 of Seq ID No 224; amino acids 23-397 of Seq ID No 235amino acids 2-420 and 414-847 of Seq ID No 240; amino acids 582-1099 ofSeq ID No 241; amino acids 1-245 of Seq ID No 242; amino acids 24-703 ofSeq ID No 247; amino acids 23-328 of Seq ID No 252; amino acids 23-248of Seq ID No 263; amino acids 2-335 of Seq ID No 267; amino acids 38-633of Seq ID No 268; amino acids 26-742 of Seq ID No 269; amino acids26-429 of Seq ID No 281; or amino acids 1-632 of Seq ID No 285. Thefragments as listed above are further defined in Seq ID Nos 188 to 203and 376, (see also Table 16).

In another embodiment, the active variant of the antigen has at least50%, especially at least 60%, preferably at least 70%, more preferablyat least 80%, still more preferably at least 90%, even more preferablyat least 95%, 96%, 97% or 98%, most preferably 99% sequence identity toamino acids 2-130 of Seq ID No 205; amino acids 26-356 of Seq ID No 216;amino acids 2-180 of Seq ID No 223; amino acids 1-168 of Seq ID No 224;amino acids 23-397 of Seq ID No 235; amino acids 2-420 and 414-847 ofSeq ID No 240; amino acids 582-1099 of Seq ID No 241; amino acids 1-245of Seq ID No 242; amino acids 24-703 of Seq ID No 247; amino acids23-328 of Seq ID No 252; amino acids 23-248 of Seq ID No 263; aminoacids 2-335 of Seq ID No 267; amino acids 38-633 of Seq ID No 268; aminoacids 26-742 of Seq ID No 269; amino acids 26-429 of Seq ID No 281; oramino acids 1-632 of Seq ID No 285.

In still another embodiment, the active variant of the antigen asdefined above is derived from the homologous sequence of a differentstrain and/or serotype of K. pneumoniae, particularly wherein theserotype is K1, K2, K3, K10, K21, K22, K30, K55, K64, O1, O2a, O3, O4,O5, or O12, or any combination of said K and said 0 serotypes.

Examples of variants of KPORF-13 (SEQ ID No 216) are given in Table 8and SEQ ID Nos 413-451. Examples of variants of KPORF-21 (SEQ ID No 224)are given in Table 9 and SEQ ID Nos 452-500. Examples of variants ofKPORF-32 (SEQ ID No 235) are given in Table 10 and SEQ ID Nos 501-540.Examples of variants of KPORF-37 (SEQ ID No 240) are given in Table 11and SEQ ID Nos 541-579. Examples of variants of KPORF-38 (SEQ ID No 241)are given in Table 12 and SEQ ID Nos 580-617. Examples of variants ofKPORF-39 (SEQ ID No 242) are given in Table 13 and SEQ ID Nos 618-667.Examples of variants of KPORF-60 (SEQ ID No 263) are given in Table 14and SEQ ID Nos 668-717. Examples of variants of KPORF-65 (SEQ ID No 268)are given in Table 15 and SEQ ID Nos 718-765.

Accordingly, in yet another embodiment of the present invention, theactive variant of the antigen as defined above is selected from thegroup consisting of SEQ ID No 413 to 765.

In still another embodiment, the antigen is further defined by

-   -   a) 1 to 400 additional amino acid residue(s), preferably 1 to        350, 1 to 300, 1 to 250, or 1 to 200, more preferably 1 to 150,        even more preferably at most 1 to 100, still more preferably at        most 1 to 50, most preferably 1, 2, 3, 4, 5, 10, 20, 30 or 40        additional amino acid residue(s) to the active fragment of the        antigen comprising or consisting of amino acids 2-420 or 414-847        of Seq ID No 240, or to the active variant of the antigen        derived from amino acids 2-420 or 414-847 of Seq ID No 240; or    -   b) 1 to 1100 additional amino acid residue(s), preferably 1 to        1000, 1 to 900, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to        400, or 1 to 300, more preferably 1 to 200, even more preferably        at most 1 to 100, still more preferably at most 1 to 50, most        preferably 1, 2, 3, 4, 5, 10, 20, 30 or 40 additional amino acid        residue(s) to the active fragment of the antigen comprising or        consisting of amino acids 582-1099 of Seq ID No 241, or to the        active variant of the antigen derived from amino acids 582-1099        of Seq ID No 241.

The additional amino acid residue(s) may be homologous to the antigen asdefined above. Homologous refers to any amino acid residue(s) whichis/are identical to the amino acid sequence of the Klebsiella antigenfrom which the fragment is derived.

Alternatively or additionally, the polypeptide may comprise or consistof the antigen, optionally the additional sequence as defined above andat least one amino acid residue heterologous to the antigen.

In an embodiment of the invention, the antigen further comprises orconsists of at least one amino acid residue heterologous to the antigen,preferably an amino acid sequence of a marker protein.

The additional sequence or amino acid residue(s) as defined aboveconsist(s) of (an) amino acid residue(s), which may be any amino acid,which may be either an L- and/or a D-amino acid, naturally occurring andotherwise. Preferably the amino acid is any naturally occurring aminoacid such as alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagine, proline, glutamine, arginine, serine, threonine,valine, tryptophan or tyrosine.

However, the amino acid may also be a modified or unusual amino acid.Examples of those are 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproicacid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyricacid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproloine,4-hydroxyproloine, isodesmosine, allo-isoleucine, N-methylglycine,N-methylisoleucine, 6-N-Methyllysine, N-methylvaline, norvaline,norleucine or ornithine. Additionally, the amino acid may be subject tomodifications such as posttranslational modifications. Examples ofmodifications include acetylation, amidation, blocking, formylation,gamma-carboxyglutamic acid hydroxylation, glycosilation, methylation,phosphorylation and sulfatation. If more than one additional orheterologous amino acid residue is present in the peptide, the aminoacid residues may be the same or different from one another.

In one embodiment the peptide of the invention further encompasses atleast one amino acid residue heterologous to the antigen. The feature“heterologous amino acid” or “amino acid heterologous to the antigen”refers to any amino acid which is different from that amino acid locatedadjacent to the antigen in any naturally occurring protein ofKlebsiellae, preferably K. pneumoniae including the three subspeciespneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K.terrigena, and K. ornithinolytica, and more preferably from K.pneumoniae or K. oxytoca. Therefore, the protein of the inventionencompassing at least one heterologous amino acid refers to a proteinwhich is different from any naturally occurring protein of Klebsiellaeor fragments thereof, preferably K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca.

In one embodiment, the additional amino acid residue(s) is/are flankingthe antigen N-terminally, C-terminally, or N- and C-terminally.

In another embodiment, the invention relates to an antigen as describedabove, whereby said additional amino acid residue(s) is/are flanking theantigen defined by

-   -   a) amino acids 2-420 of Seq ID No 240 or the variant derived        thereof C-terminally,    -   b) amino acids 414-847 of Seq ID No 240 or the variant derived        thereof N-terminally, or    -   c) amino acids 582-1099 of Seq ID No 241 or the variant derived        thereof N- and/or C-terminally.

In another embodiment, the antigen further comprises or consists ofeither a leader or a secretory sequence, a sequence employed forpurification, or a proprotein sequence.

Another aspect of the present invention relates to an antigen comprisingat least one core amino acid sequence as indicated in column “Predictedimmunogenic aa” or “Location of identified immunogenic region” of Table1, or as defined by columns “From aa” and “To aa” of Table 4, or asindicated in column “Location in protein (aa)” of Table 5, whereby morepreferably the core amino acid sequence is selected from the groupconsisting of: amino acids 11-27, 35-47, 68-107, 113-122, 124-136,140-146, 152-164, 168-174, 183-201, 211-218, 228-243, 246-253 and180-226 of Seq ID No 204; amino acids 13-31, 48-59, 69-91, 109-115,121-127 and 46-105 of Seq ID No 205; amino acids 12-44, 49-95, 102-145,148-178, 184-229, 233-244, 249-273, 292-299, 304-329, 334-348, 354-365,367-385, 394-426, 428-440, 444-487, 503-527, 531-539, 546-554, 556-584and 273-286 of Seq ID No 206; amino acids 7-17, 22-32, 34-41, 55-77,79-86, 93-111, 118-126, 131-148, 152-162, 165-177, 183-197, 213-220,234-250, 253-262, 267-294 and 211-269 of Seq ID No 207; amino acids22-29, 41-56, 58-66, 79-88, 94-121, 124-131, 134-157, 162-171, 173-180,189-197, 201-214, 216-224, 242-254, 257-270, 282-287, 290-302, 309-315,320-325, 341-355, 362-368, 372-378 and 1-48 of Seq ID No 208; aminoacids 5-15, 18-35, 48-61, 65-71, 112-119, 138-154, 157-169, 179-208,214-223, 226-232, 243-250, 256-262, 277-286, 289-296, 338-348, 352-363,370-376, 385-408, 420-436, 443-454, 462-483, 498-561, 563-592, 600-642,661-671, 673-709, 714-733, 748-754, 771-776, 798-806, 808-821, 823-839and 31-83 of Seq ID No 209; amino acids 5-14, 21-26, 31-41, 59-77,101-115, 132-145, 147-156, 180-185, 188-197 and 97-158 of Seq ID No 210;amino acids 6-18, 23-43, 45-56, 69-80, 87-97, 112-123, 135-151, 164-171,178-193, 200-227, 249-258, 262-274, 279-291, 302-308, 322-327, 329-336,351-363, 366-373, 384-399, 403-411, 415-434, 440-446, 461-482, 488-506,510-516, 518-551, 574-589, 607-629, 634-665, 667-687, 694-712, 725-739,743-751, 753-768 and 521-583 of Seq ID No 211; amino acids 4-13, 19-44,55-63, 71-82, 89-110, 120-130, 132-138, 145-161, 168-182, 189-258,261-272, 278-288, 290-301 and 11-76 of Seq ID No 212; amino acids 4-22,43-56, 63-68, 81-90, 93-99, 139-148, 155-160, 170-176, 189-195, 207-218,227-232, 241-249, 251-258, 260-266, 277-295, 300-327, 329-336, 340-356,384-390, 418-423, 427-433, 438-444 and 383-428 of Seq ID No 213; aminoacids 10-18, 32-37, 45-55, 60-69, 77-83, 89-95, 120-125, 133-170,172-185, 193-211, 214-223, 232-249, 255-275, 277-303, 305-310, 320-328,334-341, 347-353, 355-369, 380-386, 389-395 and 71-85 of Seq ID No 214;amino acids 4-23, 27-35, 67-73, 80-103, 117-126, 132-138, 140-159,162-171, 180-194, 198-208, 211-218, 228-234, 239-253, 262-270, 272-291,296-305 and 39-110 of Seq ID No 215; amino acids 13-24, 27-34, 37-66,69-88, 99-104, 149-155, 164-175, 184-193, 199-209, 227-235, 264-273,276-285, 288-315, 323-335, 346-353, 56-111 and 199-261 of Seq ID No 216;amino acids 11-22, 25-48, 51-60, 64-72, 80-96, 108-122, 132-137,142-150, 152-167, 175-199, 214-229, 237-244, 252-258, 260-266, 279-287,301-340, 345-350 and 109-153 of Seq ID No 217; amino acids 37-43, 50-57,65-82, 87-109, 123-129, 141-150, 152-157, 166-172, 179-203, 209-241,249-284, 290-300, 308-326, 329-335, 345-357, 359-368, 379-386, 390-417,420-425, 438-444, 461-466, 473-490, 497-505, 524-534, 541-550, 586-597,608-614, 622-632, 660-666, 679-694, 696-706, 708-722, 725-731, 737-763,784-789, 810-825, 837-854, 857-880, 882-895, 901-907, 911-928, 14-76 and176-220 of Seq ID No 218; amino acids 9-16, 38-52, 61-86, 93-100,110-117, 123-132, 138-145, 151-169, 172-181, 186-202, 208-225, 227-253,264-275, 289-295, 320-329, 335-342 and 113-193 of Seq ID No 219; aminoacids 11-18, 24-30, 42-49, 53-63, 69-80, 87-93, 95-103, 144-171,173-185, 193-200, 202-208, 215-221, 242-261, 266-273, 277-286, 290-299,322-328, 338-351, 354-377, 391-409, 441-451, 461-466, 499-515, 521-527,562-569, 621-629, 647-663, 676-682, 694-701, 703-713, 725-731, 735-744,755-764, 793-800 and 490-547 of Seq ID No 220; amino acids 4-11, 14-22,38-70, 81-90, 97-114, 118-132, 147-171, 173-181, 187-202, 244-250,252-298, 301-311, 313-331, 342-368, 410-418, 446-451, 456-462, 468-474,476-492, 499-507, 519-528, 552-565, 568-575, 584-613, 618-624, 626-649and 417-489 of Seq ID No 221; amino acids 4-9, 32-53, 66-72, 74-90,97-104, 110-130, 133-139, 144-152, 166-177, 203-213, 215-241, 256-275,291-304, 307-316, 321-326, 334-345, 352-367 and 201-255 of Seq ID No222; amino acids 13-19, 26-43, 66-72, 80-85, 95-101, 109-125, 131-137and 25-107 of Seq ID No 223; amino acids 13-24, 35-43, 50-56, 58-68,77-83, 104-110, 117-125, 132-138, 140-153 and 19-66 of Seq ID No 224;amino acids 15-31, 37-42, 47-54, 68-87, 89-96, 107-117, 121-127,131-137, 145-151, 176-182, 220-226, 232-246, 250-257, 291-300, 317-325,328-333, 337-359, 368-393, 403-428, 460-478, 480-493, 500-506, 511-516,519-526, 528-559, 565-572, 584-595, 597-605, 608-613, 626-648, 679-684,687-693, 703-714, 718-735, 742-750, 757-765, 768-788, 793-799, 813-819,823-829, 839-850 and 576-623 of Seq ID No 225; amino acids 10-35, 37-60,63-76, 79-86, 88-97, 108-113, 118-126, 128-134, 138-145, 153-159,168-188, 194-208, 211-243, 255-260, 270-276, 285-301, 307-346, 348-367and 275-339 of Seq ID No 226; amino acids 4-17, 21-33, 35-42, 47-64,72-80, 85-92, 98-103, 125-147, 151-161, 165-177, 183-230, 232-246,256-262, 284-306, 310-328, 331-367, 369-383, 392-399 and 32-85 of Seq IDNo 227; amino acids 5-11, 18-27, 42-52, 60-65, 75-84, 90-102, 107-116,125-178, 184-206, 221-233, 235-242, 249-257, 264-277, 288-317 and267-313 of Seq ID No 228; amino acids 5-11, 14-42, 50-75, 79-86, 89-98,120-125, 152-160, 166-181, 185-193, 200-207 and 85-114 of Seq ID No 229;amino acids 4-30, 36-43, 46-55, 63-111, 144-152, 159-168, 179-189,191-200, 205-213 and 37-109 of Seq ID No 230; amino acids 20-45, 57-77,80-100, 119-126, 131-137, 143-169, 179-185, 195-203, 207-231, 235-264,282-302, 320-329, 341-347, 353-359, 361-373 and 266-296 of Seq ID No231; amino acids 5-22, 24-37, 41-55, 57-65, 72-78, 90-103, 105-116,119-130, 164-170, 190-202, 209-231, 244-254, 260-276, 300-339, 344-350,355-376, 389-397, 399-406, 408-421, 429-437 and 103-152 of Seq ID No232; amino acids 8-16, 18-25, 31-47, 71-82, 87-102, 104-114, 126-156,176-183, 190-200, 205-212, 218-228, 231-243, 256-279, 287-301, 303-312,324-332, 335-348, 351-357, 365-380, 395-412, 422-451, 456-464, 467-483,501-507 and 405-468 of Seq ID No 233; amino acids 4-18, 21-39, 46-56,63-69, 72-86, 116-130, 132-160, 162-190, 196-201, 209-231, 233-241,251-265, 269-282, 292-298, 309-324, 333-369, 391-415, 417-427, 436-454,471-480, 482-499, 510-518, 521-533, 537-543, 545-561, 571-581, 585-597,599-607, 609-635, 638-643, 650-665, 671-685, 687-695, 701-707, 710-720,724-736, 747-757, 764-769, 772-784, 791-796, 808-820 and 317-401 of SeqID No 234; amino acids 4-12, 15-33, 58-77, 82-89, 98-106, 108-118,120-135, 141-147, 152-160, 168-215, 225-233, 235-247, 250-264, 284-312,314-321, 336-343, 359-374, 386-394 and 159-218 of Seq ID No 235; aminoacids 4-16, 24-36, 40-47, 49-56, 61-81, 84-143, 148-156, 158-164,170-175, 194-206, 208-214 and 126-203 of Seq ID No 236; amino acids28-45, 50-61, 94-111, 113-124, 137-142, 147-173, 180-188, 190-196,202-223, 229-235, 239-249, 262-270, 280-288, 290-321, 325-332, 347-355,359-368, 389-407, 415-427, 429-453, 458-465, 477-485, 499-505, 516-527,531-549, 569-592, 594-602, 605-615, 628-635, 647-659, 662-683, 727-735,760-765, 771-780, 788-809, 811-818 and 549-630 of Seq ID No 237; aminoacids 21-28, 33-40, 48-100, 104-111, 113-134 and 1-46 of Seq ID No 238;amino acids 12-24, 31-41, 53-61, 73-87, 112-128, 133-140, 151-156 and26-98 of Seq ID No 239; amino acids 4-9, 19-26, 32-56, 58-67, 71-81,90-95, 97-105, 112-118, 124-132, 138-144, 147-167, 169-177, 199-207,212-217, 231-241, 250-260, 266-272, 274-282, 289-296, 299-310, 316-331,344-350, 352-363, 368-377, 381-394, 399-406, 412-450, 459-473, 486-503,508-514, 518-548, 564-570, 579-587, 602-608, 616-623, 628-635, 638-654,678-688, 691-696, 703-709, 716-723, 761-772, 784-793, 819-826, 835-844and 790-834 of Seq ID No 240; amino acids 4-10, 18-36, 43-50, 63-71,75-105, 109-117, 134-140, 145-157, 176-182, 184-201, 203-211, 215-225,240-250, 262-284, 294-309, 313-319, 327-337, 350-356, 361-367, 372-393,411-421, 428-451, 453-466, 487-492, 501-528, 535-553, 564-574, 592-605,612-629, 631-640, 646-653, 658-666, 673-681, 713-718, 720-730, 739-749,784-792, 821-826, 833-844, 853-863, 871-876, 885-894, 900-918, 937-950,952-957, 972-990, 995-1001, 1024-1036, 1039-1044, 1049-1055, 1062-1089,1091-1103, 1110-1121, 1123-1129, 1131-1151, 1157-1179, 1181-1201,1204-1223, 1233-1244, 1269-1276, 1279-1286, 1294-1301, 1303-1309,1315-1338, 1350-1362, 1373-1381, 1398-1406, 1412-1423, 1440-1446,1458-1466, 1481-1487, 1492-1508, 1511-1518, 1528-1534, 1536-1547,1553-1565, 1606-1617, 1619-1644 and 761-781 of Seq ID No 241; aminoacids 6-13, 31-38, 47-60, 71-102, 107-123, 128-155, 173-179, 185-194,210-220 and 161-232 of Seq ID No 242; amino acids 11-34, 36-43, 49-67,74-79, 84-92, 94-100, 103-112, 120-129, 134-155, 162-173, 177-185,189-202, 206-211 and 130-185 of Seq ID No 243; amino acids 4-10, 20-35,37-46, 48-55, 60-66, 75-82, 87-98, 133-150, 166-172, 178-189, 208-214,230-235, 245-251, 271-308, 319-333, 335-355, 373-380 and 117-201 of SeqID No 244; amino acids 4-30, 54-65, 91-105, 107-131, 135-154, 163-192,199-208, 210-224, 229-239, 248-257, 263-279, 281-294, 328-354, 373-379,382-405, 426-453, 462-487 and 249-323 of Seq ID No 245; amino acids4-10, 12-24, 45-55, 75-88 and 24-40 of Seq ID No 246; amino acids 4-14,20-37, 47-53, 55-61, 75-81, 97-103, 107-124, 129-135, 139-147, 160-166,169-175, 181-190, 202-221, 247-255, 272-285, 300-310, 318-332, 351-361,384-397, 406-427, 442-449, 458-482, 494-503, 512-524, 531-539, 552-562,577-588, 590-596, 600-608, 613-624, 637-668, 692-700 and 232-278 of SeqID No 247; amino acids 33-39, 49-55, 68-84, 90-96, 104-120, 126-143,150-159, 168-191, 197-208, 219-225, 227-233, 241-247, 63-115 and 200-250of Seq ID No 248; amino acids 4-22, 24-34, 36-55, 57-76, 83-97, 99-117,135-143, 145-157, 163-174, 178-198, 200-207, 209-270, 276-290, 321-335,338-347, 367-374, 393-402, 404-411, 416-422, 443-460, 467-473 and117-183 of Seq ID No 249; amino acids 26-37, 44-52, 57-96, 104-111,118-124, 155-177, 179-197, 201-214, 223-233, 243-250, 257-262, 291-297,303-314, 319-363 and 47-105 of Seq ID No 250; amino acids 36-43, 45-60,76-97, 107-125, 131-156, 158-164 and 118-163 of Seq ID No 251; aminoacids 5-32, 40-50, 52-60, 70-88, 92-101, 106-126, 138-150, 152-161,175-193, 201-234, 237-248, 270-285, 297-303, 312-318 and 209-255 of SeqID No 252; amino acids 4-12, 23-34, 49-55, 59-65, 70-81, 83-130 and62-113 of Seq ID No 253; amino acids 4-26, 38-49, 69-76, 82-96, 103-119,126-140, 143-190, 194-209, 212-218 and 100-167 of Seq ID No 254; aminoacids 7-29, 35-47, 56-66, 80-94, 97-123, 125-148, 150-160, 166-173,175-191, 193-200, 207-225 and 75-176 of Seq ID No 255; amino acids14-36, 39-45, 51-59, 66-71, 76-88, 106-117, 121-126, 140-157, 164-187,198-206, 210-252 and 202-256 of Seq ID No 256; amino acids 4-19, 27-35,90-107, 120-134, 144-150, 166-175, 192-198, 221-243, 249-255, 263-278,283-288, 305-321, 324-334, 342-349, 355-366, 377-390, 413-425, 442-448and 130-178 of Seq ID No 257; amino acids 17-26, 41-51, 54-61, 64-72,78-105, 117-125, 127-137, 147-155, 175-213, 230-236, 238-261, 271-277,282-297, 309-318, 329-347, 355-372, 377-390 and 69-126 of Seq ID No 258;amino acids 4-48, 54-60, 62-69, 73-81, 88-115, 124-137, 139-154,156-169, 171-190, 194-231, 240-273, 288-303, 336-363, 367-395, 405-411,434-442, 449-454, 466-483, 491-507 and 226-282 of Seq ID No 259; aminoacids 26-34, 39-47, 50-80, 82-88, 97-105, 108-127, 131-137, 162-180,185-191, 198-203, 209-214, 226-247, 256-288, 296-305 and 149-239 of SeqID No 260; amino acids 5-28, 30-54, 73-84, 89-98, 109-116, 122-128,137-142, 163-189, 207-236, 245-280, 288-390, 404-423, 426-433, 450-474,487-504, 506-513, 524-530, 532-595, 605-614, 620-626, 631-638, 644-657,667-683, 686-693, 695-702, 707-733, 739-747 and 6-62 of Seq ID No 261;amino acids 23-31, 39-50, 55-67, 76-100, 117-130, 149-171, 173-185,218-238, 242-288, 291-298, 334-346, 355-369, 382-399, 413-420, 431-438,442-449, 455-466, 486-493, 498-508, 524-531, 540-546, 551-558, 562-570,575-582, 585-596, 598-604, 621-630, 632-650, 670-677, 682-701, 736-749,755-761 and 612-626 of Seq ID No 262; amino acids 4-21, 24-39, 44-68,74-81, 85-91, 109-116, 129-138, 142-148, 173-188, 195-201, 207-212,223-228 and 126-148 of Seq ID No 263; amino acids 4-17, 24-42, 61-67,84-93, 96-102, 116-121, 135-143, 155-165, 177-186, 210-224, 253-259,272-297, 299-331, 337-351, 359-367, 369-385 and 1-49 of Seq ID No 264;amino acids 4-25, 28-54, 67-81, 85-136, 138-143, 157-170, 180-190,197-203, 205-214, 219-243, 246-270, 277-283, 290-299, 305-311 and127-182 of Seq ID No 265; amino acids 11-20, 25-33, 75-80, 85-91,113-124, 143-155, 161-170, 172-184 and 128-176 of Seq ID No 266; aminoacids 4-9, 16-26, 28-34, 55-80, 120-143, 150-156, 158-164, 167-178,185-190, 192-213, 221-237, 242-255, 257-272, 281-290, 325-332 and 48-106of Seq ID No 267; amino acids 13-48, 59-70, 78-88, 95-112, 129-151,153-161, 163-182, 214-221, 235-245, 248-277, 281-291, 293-301, 303-311,315-320, 323-346, 377-383, 390-398, 447-454, 474-487, 491-512, 531-544,547-553, 582-590, 597-603, 605-611, 623-629 and 410-466 of Seq ID No268; amino acids 6-26, 39-46, 48-58, 69-75, 109-121, 139-144, 148-155,166-172, 215-221, 261-267, 313-319, 363-386, 423-433, 447-458, 465-471,483-494, 497-517, 558-565, 578-586, 589-597, 619-626, 636-645, 659-665,671-680, 682-693, 733-739 and 152-206 of Seq ID No 269; amino acids4-19, 23-35, 40-50, 52-58, 65-73, 78-103, 112-125, 146-160, 163-192,194-200 and 29-90 of Seq ID No 270; amino acids 4-13, 17-32, 40-50,57-67, 76-81, 88-95, 107-119, 131-142, 144-157, 171-178, 185-193,197-207, 212-227, 231-238, 248-253, 263-310 and 90-170 of Seq ID No 271;amino acids 9-28, 57-82, 84-93, 126-135, 143-166, 173-194, 196-201,212-220, 228-254, 269-277, 289-298, 305-316, 320-327, 330-337, 350-359,373-378, 386-392, 403-411, 421-428, 435-441, 443-458, 465-470 and 80-141of Seq ID No 272; amino acids 11-48, 54-67, 69-75, 89-95, 101-122,124-131, 134-157, 159-175, 202-208, 214-228, 258-270, 272-280, 287-295,298-310, 331-338, 340-417, 427-500, 502-509, 534-552, 556-561, 564-577,585-592, 594-608, 621-627, 632-641, 643-652, 671-681, 683-709, 712-743,758-764, 776-783, 789-820, 835-851, 864-883, 885-910, 913-940, 948-953,967-976, 994-1020 and 775-825 of Seq ID No 273; amino acids 14-24,32-54, 58-63, 70-80, 93-100, 108-125, 127-135, 142-153, 155-160,180-191, 201-208, 210-216, 222-235, 242-264, 267-273, 276-282, 284-308and 10-59 of Seq ID No 274; amino acids 16-28, 44-68, 70-77, 83-90,99-129, 131-137, 145-154, 161-175, 183-190, 196-203, 205-220, 238-245,321-328, 330-338, 366-379, 383-397, 399-405, 412-418, 442-458, 471-483,486-505, 536-544, 562-568, 583-602, 610-618, 629-635, 641-655, 672-682,697-705, 714-729, 744-751, 755-762, 766-771, 783-807 and 555-621 of SeqID No 275; amino acids 4-9, 20-34, 45-54, 60-77, 79-89, 91-100, 102-149,162-170, 177-189, 193-208, 210-222, 238-244, 252-264, 267-276, 302-307and 100-140 of Seq ID No 276; amino acids 11-27, 30-49, 56-62, 69-74,76-85, 94-108, 116-125, 129-147, 153-161, 165-171, 177-208, 217-223,225-231, 237-255, 260-284, 293-300 and 73-137 of Seq ID No 277; aminoacids 4-38, 40-51, 84-97, 99-106, 109-115, 119-129, 131-145, 148-160,180-186, 188-202, 230-243, 246-267, 274-288, 290-299, 302-312, 317-327,332-344, 353-377, 381-388, 407-419, 423-437, 447-470, 474-482, 486-494,501-523, 531-546, 551-556 and 727-740 of Seq ID No 278; amino acids23-52, 62-76, 87-104, 109-115, 117-123, 129-139, 143-149, 152-170,172-191, 199-205, 212-218, 220-240, 249-256, 263-275, 297-303, 308-342,349-380, 382-394, 414-420, 430-441, 446-452, 460-475, 488-505, 514-531,533-539, 546-568, 570-577, 579-588, 613-625, 632-670, 672-716, 718-745,759-769, 785-798, 801-807 and 272-324 of Seq ID No 279; amino acids4-34, 36-43, 56-73, 80-87, 101-134, 148-159, 161-170, 178-185, 195-206,211-221, 223-248, 259-271, 276-295, 297-308 and 241-296 of Seq ID No280; amino acids 5-31, 44-50, 64-74, 86-94, 132-147, 154-167, 196-203,209-219, 253-260, 284-289, 300-312, 319-327, 335-340, 358-364, 376-383and 166-202 of Seq ID No 281; amino acids 4-9, 12-27, 29-71, 77-84,90-108, 114-142, 147-164, 180-213, 217-227, 229-282, 291-309, 322-329,336-353, 365-370 and 317-364 of Seq ID No 282; amino acids 36-41, 52-66,71-83, 89-95, 116-127, 154-174, 176-184, 200-206, 230-237, 248-259,269-284, 307-316, 376-383, 399-418, 424-442, 445-451, 454-462 and 1-50of Seq ID No 283; amino acids 9-14, 33-49, 64-72, 87-92, 103-109,123-128, 130-141, 143-154, 160-166, 182-214, 237-247, 251-260, 292-300,327-332, 337-350, 357-365, 388-398, 405-411, 422-428, 451-459, 478-488,520-531, 534-540, 558-564, 580-586, 591-600, 605-615, 629-635, 641-653,658-672, 212-244 and 533-611 of Seq ID No 284; amino acids 4-10, 17-27,30-37, 44-62, 80-85, 94-114, 118-131, 134-141, 148-161, 171-212,218-241, 248-261, 274-313, 325-336, 342-348, 359-373, 391-397, 424-431,454-474, 489-495, 497-503, 505-515, 548-553, 560-580, 591-610 and277-324 of Seq ID No 285; amino acids 7-16, 18-24, 30-47, 49-70, 83-99,103-117, 126-141, 146-153, 159-165, 177-194, 198-221, 236-246, 255-262,273-279, 283-296, 301-332, 338-411, 422-428, 434-440, 452-458, 463-469,494-509, 511-517, 524-531, 548-554, 564-572 and 335-389 of Seq ID No286; amino acids 9-15, 33-54, 56-80, 102-108 and 1-42 of Seq ID No 287;amino acids 15-36, 42-55, 58-68 and 54-77 of Seq ID No 288; amino acids55-75, 89-96, 98-110 and 14-36 of Seq ID No 289; amino acids 8-14,29-51, 73-101, 110-117 and 70-114 of Seq ID No 290; amino acids 20-25,29-34, 41-52, 60-67, 69-85, 90-100, 114-122, 136-142, 160-170, 174-181and 21-58 of Seq ID No 291; amino acids 14-22 and 4-13 of Seq ID No 292;amino acids 22-40, 54-66, 88-105, 109-118 and 31-74 of Seq ID No 293;amino acids 5-11, 18-32, 47-60, 66-73, 83-92, 113-120, 126-141, 151-164,167-174, 201-211 and 118-129 of Seq ID No 294; amino acids 5-11, 18-24,32-40, 47-53 and 25-54 of Seq ID No 295; amino acids 18-24, 31-48 and5-55 of Seq ID No 296; amino acids 10-16, 26-32, 47-56, 85-95 and 10-62of Seq ID No 297; amino acids 4-12, 16-26 and 25-34 of Seq ID No 298;amino acids 19-29, 45-51, 63-68, 76-92, 103-110, 114-120, 123-133,135-141 and 14-78 of Seq ID No 299; amino acids 4-18, 47-61 and 57-93 ofSeq ID No 300; amino acids 17-29, 44-50 and 26-38 of Seq ID No 301;amino acids 5-19, 55-64, 78-85, 95-101, 104-112 and 24-33 of Seq ID No302; amino acids 4-10 and 12-31 of Seq ID No 303; amino acids 4-12,27-41, 43-58, 60-67, 76-86 and 13-65 of Seq ID No 304; amino acids30-38, 57-67 and 5-32 of Seq ID No 305; amino acids 30-43 and 2-21 ofSeq ID No 306; amino acids 14-20, 23-36, 41-48 and 1-52 of Seq ID No307; amino acids 18-33, 51-58, 76-82 and 32-46 of Seq ID No 308; aminoacids 25-31 and 2-16 of Seq ID No 309; amino acids 14-23, 50-58 and 9-49of Seq ID No 310; amino acids 4-10, 22-31, 35-45, 48-68, 71-80 and 17-66of Seq ID No 311; amino acids 4-24, 28-42, 46-56, 63-69, 87-94, 112-131and 2-46 of Seq ID No 312; amino acids 4-15, 19-28, 34-41, 52-62, 78-86and 2-20 of Seq ID No 313; amino acids 4-11, 16-30, 32-42 and 7-38 ofSeq ID No 314; amino acids 4-20, 22-31 and 22-38 of Seq ID No 315; aminoacids 4-19 and 17-32 of Seq ID No 316; amino acids 7-13, 17-22, 27-33,80-100 and 26-40 of Seq ID No 317; amino acids 10-18, 22-48 and 32-44 ofSeq ID No 318; amino acids 15-24, 43-49, 73-83 and 45-93 of Seq ID No319; amino acids 22-29, 46-55, 57-63 and 5-17 of Seq ID No 320; aminoacids 10-33 and 21-35 of Seq ID No 321; amino acids 16-24 and 22-49 ofSeq ID No 322; amino acids 4-16, 37-73, 76-110, 117-125, 127-132 and2-30 of Seq ID No 323; amino acids 4-12, 23-35, 44-56, 59-88 and 22-76of Seq ID No 324; amino acids 15-26 and 23-35 of Seq ID No 325; aminoacids 12-22, 31-40 and 17-44 of Seq ID No 326; amino acids 4-9, 13-18,29-35 and 57-64 of Seq ID No 327; amino acids 31-55, 67-81 and 25-70 ofSeq ID No 328; amino acids 13-24, 51-58 and 13-26 of Seq ID No 329;amino acids 6-20, 29-40, 57-79 and 46-88 of Seq ID No 330; amino acids8-14, 41-54, 68-76, 83-93, 106-126, 130-139 and 12-72 of Seq ID No 331;amino acids 5-13, 17-24, 41-55, 64-69, 80-85, 94-107, 109-115 and 53-88of Seq ID No 332; amino acids 5-12, 32-54, 57-64 and 20-33 of Seq ID No333; amino acids 4-16, 40-48, 50-58, 62-68, 75-85, 92-104, 108-116,124-134 and 68-128 of Seq ID No 334; amino acids 7-13, 19-29, 34-40,54-71, 76-81, 91-144, 147-155, 157-188 and 11-83 of Seq ID No 335; aminoacids 17-24, 32-41 and 6-43 of Seq ID No 336; amino acids 14-31, 38-59,69-87, 95-102, 126-146, 157-162, 177-193, 201-227, 238-251 and 63-78 ofSeq ID No 337; amino acids 10-16, 18-25, 27-41, 43-52, 59-86, 94-101,134-140 and 38-100 of Seq ID No 338; amino acids 4-19, 23-35, 43-72,78-92 and 37-93 of Seq ID No 339; amino acids 15-20, 27-32, 41-65,69-82, 93-105, 107-115, 120-147, 170-178, 184-201, 214-257, 272-281,293-314, 332-339, 358-364, 374-381, 390-397, 399-414, 428-460 and317-375 of Seq ID No 340; amino acids 11-28, 47-55, 59-68, 76-105,108-116, 120-144, 146-160, 167-175, 180-187, 209-233 and 144-158 of SeqID No 341; amino acids 4-13, 58-78 and 14-77 of Seq ID No 342; aminoacids 26-31, 44-49, 57-64, 67-74, 107-112, 116-152, 154-181, 202-212,241-255 and 57-101 of Seq ID No 343; amino acids 10-41, 53-70, 81-93,100-111, 137-147, 164-169, 183-190, 199-210, 216-221, 226-240 and 84-95of Seq ID No 344; amino acids 12-45, 48-56, 73-79, 91-103, 106-112,117-125, 132-143, 154-160, 178-201, 208-214, 216-225, 260-266, 276-283and 98-115 of Seq ID No 345; amino acids 4-15, 30-42 and 29-39 of Seq IDNo 346; amino acids 22-53, 55-73, 80-88 and 33-66 of Seq ID No 347;amino acids 6-23, 44-54 and 56-67 of Seq ID No 348; amino acids 8-21,35-44, 66-75, 82-87, 94-101 and 32-94 of Seq ID No 349; amino acids8-20, 23-32, 36-50, 53-69 and 15-69 of Seq ID No 350; amino acids 8-22of Seq ID No 351; amino acids 31-37 and 2-31 of Seq ID No 352; aminoacids 4-20, 23-39, 58-63, 71-78, 97-102 and 22-82 of Seq ID No 353;amino acids 23-44, 135-152, 168-184 and 57-116 of Seq ID No 354; aminoacids 24-31, 42-50, 52-62, 93-117 and 43-94 of Seq ID No 355; aminoacids 20-29 and 24-43 of Seq ID No 356; amino acids 12-57, 59-74 and22-40 of Seq ID No 357; amino acids 7-16, 18-26, 39-45, 68-78, 86-92 and65-82 of Seq ID No 358; amino acids 5-17, 19-34, 42-48, 56-71, 102-113,118-129 and 67-111 of Seq ID No 359; amino acids 4-33, 50-71 and 13-55of Seq ID No 360; amino acids 9-17, 23-30, 37-54, 69-88, 96-102,114-123, 130-140, 143-163 and 5-70 of Seq ID No 361; amino acids 4-23,27-52, 71-80 and 9-94 of Seq ID No 362; amino acids 13-19 and 2-21 ofSeq ID No 363; amino acids 18-26, 28-52, 63-74, 94-107, 123-134 and18-84 of Seq ID No 364; amino acids 19-33, 57-68 and 26-48 of Seq ID No365; amino acids 4-26, 31-37, 42-59 and 12-65 of Seq ID No 366; aminoacids 4-25 and 20-39 of Seq ID No 367; amino acids 40-51, 54-62, 67-75,83-89, 126-146, 148-156 and 31-42 of Seq ID No 368; amino acids 4-15,23-33, 38-49, 82-98 and 7-91 of Seq ID No 369; amino acids 6-26, 36-57and 40-64 of Seq ID No 370; amino acids 6-15, 21-28, 32-38, 57-65,78-103, 114-134, 138-144, 154-163 and 41-95 of Seq ID No 371; aminoacids 13-30, 47-57, 71-76 and 25-71 of Seq ID No 372; amino acids 4-31,43-51, 55-63, 67-72, 76-83, 88-95, 99-118, 125-132, 134-159 and 82-118of Seq ID No 373; amino acids 4-17, 26-32, 34-40, 45-61, 67-92 and 41-97of Seq ID No 374; amino acids 179-208 and 198-227 of Seq ID No 204;amino acids 45-69, 65-89 and 83-106 of Seq ID No 205; amino acids269-290 of Seq ID No 206; amino acids 209-230, 226-249 and 245-269 ofSeq ID No 207; amino acids-9-15, 10-33 and 28-52 of Seq ID No 208; aminoacids 29-50, 45-67 and 62-85 of Seq ID No 209; amino acids 96-120,115-139 and 134-158 of Seq ID No 210; amino acids 519-543, 539-563 and559-584 of Seq ID No 211; amino acids 10-35, 31-56 and 52-77 of Seq IDNo 212; amino acids 382-407 and 403-428 of Seq ID No 213; amino acids66-90 of Seq ID No 214; amino acids 38-65 and 61-88 of Seq ID No 215;amino acids 56-85, 198-221, 217-240 and 236-261 of Seq ID No 216; aminoacids 108-132 and 128-153 of Seq ID No 217; amino acids 13-37, 33-56,52-76, 175-200 and 196-220 of Seq ID No 218; amino acids 132-156,152-176 and 172-195 of Seq ID No 219; amino acids 489-512, 508-531 and526-549 of Seq ID No 220; amino acids 416-442, 438-465 and 461-489 ofSeq ID No 221; amino acids 199-222, 217-240 and 235-257 of Seq ID No222; amino acids 25-55, 51-81 and 77-107 of Seq ID No 223; amino acids18-46 and 42-66 of Seq ID No 224; amino acids 575-601 and 597-623 of SeqID No 225; amino acids 274-299, 295-320 and 316-339 of Seq ID No 226;amino acids 32-61 and 57-85 of Seq ID No 227; amino acids 266-291 and287-313 of Seq ID No 228; amino acids 85-114 of Seq ID No 229; aminoacids 36-64 and 83-109 of Seq ID No 230; amino acids 264-285 and 280-300of Seq ID No 231; amino acids 102-128 and 124-152 of Seq ID No 232;amino acids 404-429 and 445-468 of Seq ID No 233; amino acids 343-374and 370-401 of Seq ID No 234; amino acids 158-182 and 178-202 of Seq IDNo 235; amino acids 151-180 of Seq ID No 236; amino acids 549-579,575-605 and 601-630 of Seq ID No 237; amino acids-7-23 and 19-46 of SeqID No 238; amino acids 48-75 and 71-98 of Seq ID No 239; amino acids789-813 and 809-834 of Seq ID No 240; amino acids 759-783 of Seq ID No241; amino acids 160-188, 184-211 and 207-232 of Seq ID No 242; aminoacids 130-159 of Seq ID No 243; amino acids 117-147, 143-173 and 169-201of Seq ID No 244; amino acids 248-276, 272-300 and 296-323 of Seq ID No245; amino acids 21-43 of Seq ID No 246; amino acids 231-256 and 252-278of Seq ID No 247; amino acids 62-91, 87-115 and 199-227 of Seq ID No248; amino acids 116-141, 137-162 and 158-183 of Seq ID No 249; aminoacids 46-69, 65-87 and 82-105 of Seq ID No 250; amino acids 117-142 and138-163 of Seq ID No 251; amino acids 208-233 and 229-255 of Seq ID No252; amino acids 61-88 of Seq ID No 253; amino acids 99-124, 120-145 and141-167 of Seq ID No 254; amino acids 74-103, 99-128, 124-152 and148-176 of Seq ID No 255; amino acids 202-231 and 227-256 of Seq ID No256; amino acids 129-154 and 150-178 of Seq ID No 257; amino acids95-126 of Seq ID No 258; amino acids 226-256 and 252-282 of Seq ID No259; amino acids 171-198, 194-221 and 217-240 of Seq ID No 260; aminoacids 35-65 and 61-91 of Seq ID No 261; amino acids 608-631 of Seq ID No262; amino acids 124-149 of Seq ID No 263; amino acids-14-21 and 17-49of Seq ID No 264; amino acids 127-157 and 153-182 of Seq ID No 265;amino acids 150-176 of Seq ID No 266; amino acids 48-79 and 75-106 ofSeq ID No 267; amino acids 435-466 of Seq ID No 268; amino acids 151-180and 176-206 of Seq ID No 269; amino acids 126-151 and 167-190 of Seq IDNo 270; amino acids 89-118, 114-144 and 140-170 of Seq ID No 271; aminoacids 80-112 of Seq ID No 272; amino acids 9-36 of Seq ID No 274; aminoacids 117-140 of Seq ID No 276; amino acids 72-97, 93-117 and 113-137 ofSeq ID No 277; amino acids 723-746 of Seq ID No 278; amino acids 271-300of Seq ID No 279; amino acids 240-271 and 267-296 of Seq ID No 280;amino acids 165-188 and 183-206 of Seq ID No 281; amino acids 316-344and 340-364 of Seq ID No 282; amino acids-3-27 and 23-50 of Seq ID No283; amino acids 212-244, 532-561, 557-586 and 582-611 of Seq ID No 284;amino acids 276-302 and 298-324 of Seq ID No 285; amino acids 335-364and 360-389 of Seq ID No 286; amino acids 41-64 and 59-82 of Seq ID No287; amino acids 53-77 of Seq ID No 288; amino acids 13-37 of Seq ID No289; amino acids 69-94 and 90-114 of Seq ID No 290; amino acids 19-42and 37-60 of Seq ID No 291; amino acids 1-25 of Seq ID No 292; aminoacids 30-54 and 50-75 of Seq ID No 293; amino acids 111-135 of Seq ID No294; amino acids 25-54 of Seq ID No 295; amino acids 67-98 and 94-126 ofSeq ID No 334; amino acids 9-32, 27-51, 46-70 and 65-86 of Seq ID No335; amino acids 1-24 and 20-44 of Seq ID No 336; amino acids 58-82 ofSeq ID No 337; amino acids 37-62, 58-82 and 77-101 of Seq ID No 338;amino acids 37-68 and 64-93 of Seq ID No 339; amino acids 317-347 and343-375 of Seq ID No 340; amino acids 140-164 of Seq ID No 341; aminoacids 13-40, 36-60 and 55-79 of Seq ID No 342; amino acids 56-79 and75-101 of Seq ID No 343; amino acids 77-101 of Seq ID No 344; aminoacids 94-118 of Seq ID No 345; amino acids 46-105 of Seq ID No 205;amino acids 56-111 of Seq ID No 216; amino acids 25-107 of Seq ID No223; amino acids 19-66 of Seq ID No 224; amino acids 85-114 of Seq ID No229; amino acids 37-109 of Seq ID No 230; amino acids 266-296 of Seq IDNo 231; amino acids 103-152 of Seq ID No 232; amino acids 167-218 of SeqID No 235; amino acids 790-834 of Seq ID No 240; amino acids 761-781 ofSeq ID No 241; amino acids 176-232 of Seq ID No 242; amino acids 117-201of Seq ID No 244; amino acids 249-323 of Seq ID No 245; amino acids232-278 of Seq ID No 247; amino acids 209-255 of Seq ID No 252; aminoacids 75-176 of Seq ID No 255; amino acids 202-256 of Seq ID No 256;amino acids 130-178 of Seq ID No 257; amino acids 69-126 of Seq ID No258; amino acids 126-148 of Seq ID No 263; amino acids 1-49 of Seq ID No264; amino acids 127-182 of Seq ID No 265; amino acids 48-106 of Seq IDNo 267; amino acids 410-466 of Seq ID No 268; amino acids 152-206 of SeqID No 269; amino acids 555-621 of Seq ID No 275; amino acids 166-202 ofSeq ID No 281; amino acids 317-364 of Seq ID No 282; amino acids 1-50 ofSeq ID No 283; amino acids 277-324 of Seq ID No 285; amino acids 14-36of Seq ID No 289; amino acids 6-43 of Seq ID No 336; amino acids 57-101of Seq ID No 343; amino acids 84-95 of Seq ID No 344; amino acids 98-115of Seq ID No 345.

In one embodiment the antigen further consists of

-   -   a) 1 to 50 additional amino acid residue(s), preferably 1 to 40,        more preferably 1 to 30, even more preferably at most 1 to 25,        still more preferably at most 1 to 10, most preferably 1, 2, 3,        4 or 5 additional amino acid residue(s); and/or    -   b) at least one amino acid residue heterologous to the core        amino acid sequence.

Said additional amino acid residue(s) are further defined above.

In another embodiment said amino acid residue(s) is/are flanking thecore amino acid sequence N-terminally, C-terminally, or N- andC-terminally.

In an embodiment of the invention the antigen comprises at least 2, atleast 3, at least 4, at least 5 or at least 6 core amino acid sequencesas defined above.

The problem underlying the present invention is solved in another aspectby a process for producing an antigen, or an active fragment or anactive variant thereof, as defined in the present invention, comprisingexpressing the nucleic acid molecule as defined above.

The present invention further relates to a process for producing a cellwhich expresses an antigen, or an active fragment or an active variantthereof, as defined above, comprising transforming or transfecting asuitable host cell with the vector as defined above.

In an embodiment, the antigen, or the active fragment or the activevariant thereof, is isolated from Klebsiella, preferably K. pneumoniaeincluding the three subspecies pneumoniae, ozaenae and rhinoscleromatis,K. oxytoca, K. planticola, K. terrigena, and K. ornithinolytica, andmore preferably from K. pneumoniae or K. oxytoca.

The problem underlying the present invention is solved in another aspectby a pharmaceutical composition, preferably a vaccine, comprising anantigen, or an active fragment or an active variant thereof, as definedabove, or a nucleic acid molecule as defined above or a vector asdefined above.

Another aspect of the present invention provides a pharmaceuticalcomposition, preferably a vaccine, comprising an antigen, or an activefragment or an active variant thereof, as defined above, or a nucleicacid molecule as defined above or a vector as defined above for thetreatment or prevention of an infection with Klebsiella, preferably K.pneumoniae including the three subspecies pneumoniae, ozaenae andrhinoscleromatis, K. oxytoca, K. planticola, K. terrigena, and K.ornithinolytica, and more preferably from K. pneumoniae or K. oxytoca.

In a preferred embodiment the pharmaceutical composition of the presentinvention further comprises an immunostimulatory substance, preferablyselected from the group comprising polycationic polymers, especiallypolycationic peptides, immunostimulatory oligo-deoxynucleotides (ODNs),especially Oligo(dIdC)₁₃, peptides containing at least two LysLeuLysmotifs, especially KLKLLLLLKLK, neuroactive compounds, especially humangrowth hormone, alum, Freund's complete or incomplete adjuvants, orcombinations thereof.

In a more preferred embodiment of the pharmaceutical composition of thepresent invention the immunostimulatory substance is a combination ofeither a polycationic polymer and immunostimulatory deoxynucleotides orof a peptide containing at least two LysLeuLys motifs andimmunostimulatory deoxynucleotides, preferably a combination ofKLKLLLLLKLK and Oligo(dIdC)₁₃.

In a still more preferred embodiment of the pharmaceutical compositionof the present invention the polycationic polymer is a polycationicpeptide, especially polyarginine.

Still another aspect of the present invention provides an antigen, or anactive fragment or an active variant thereof, as defined above, or anucleic acid molecule as defined above or a vector as defined above forthe treatment or prevention of an infection with Klebsiella, preferablyK. pneumoniae including the three subspecies pneumoniae, ozaenae andrhinoscleromatis, K. oxytoca, K. planticola, K. terrigena, and K.ornithinolytica, and more preferably from K. pneumoniae or K. oxytoca.

Another preferred embodiment of the invention relates to the use of anucleic acid molecule as defined above, or an antigen, an activefragment or an active variant thereof, as defined above, for thepreparation of a pharmaceutical composition, especially for thepreparation of a vaccine, for treating or preventing infections withKlebsiella, preferably K. pneumoniae including the three subspeciespneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K.terrigena, and K. ornithinolytica, and more preferably from K.pneumoniae or K. oxytoca.

The problem underlying the present invention is solved in a furtheraspect by an antibody, or at least an effective part thereof, whichbinds to at least a selective part of an antigen, or a fragment thereof,preferably an active fragment thereof, or a variant thereof, preferablyan active variant thereof, as defined above.

In a preferred embodiment the antibody is a monoclonal antibody.

In another preferred embodiment said effective part comprises a Fabfragment, a F(ab) fragment, a F(ab) N fragment, a F (ab)₂ fragment or aF_(v) fragment.

In still another embodiment of the invention the antibody is a chimericantibody.

In yet another embodiment the antibody is a humanized antibody.

Another aspect of the invention relates to a hybridoma cell line, whichproduces an antibody as defined above.

The problem underlying the present invention is furthermore solved by amethod for producing an antibody as defined above, characterized by thefollowing steps:

-   -   a) initiating an immune response in a non-human animal by        administrating an antigen, or an active fragment or an active        variant thereof, as defined above, to said animal,    -   b) removing an antibody containing body fluid from said animal,        and    -   c) producing the antibody by subjecting said antibody containing        body fluid to further purification steps.

The invention further relates to a method for producing an antibody asdefined above, characterized by the following steps:

-   -   a) initiating an immune response in a non-human animal by        administrating an antigen, or an active fragment or an active        variant thereof, as defined above, to said animal,    -   b) removing the spleen or spleen cells from said animal,    -   c) producing hybridoma cells of said spleen or spleen cells,    -   d) selecting and cloning hybridoma cells specific for said        antigen, or for said active fragment or for said active variant        thereof,    -   e) producing the antibody by cultivation of said cloned        hybridoma cells, and    -   f) optionally conducting further purification steps.

Another aspect of the present invention is related to a pharmaceuticalcomposition comprising an antibody as specified above.

Still another aspect relates to an antibody as defined above or apharmaceutical composition comprising an antibody as defined above forthe treatment or prevention of an infection with Klebsiella, preferablyK. pneumoniae including the three subspecies pneumoniae, ozaenae andrhinoscleromatis, K. oxytoca, K. planticola, K. terrigena, and K.ornithinolytica, and more preferably from K. pneumoniae or K. oxytoca.

The problem underlying the present invention is solved in another aspectby the use of an antibody as defined above for the preparation of apharmaceutical composition for treating or preventing infections withKlebsiella, preferably K. pneumoniae including the three subspeciespneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K.terrigena, and K. ornithinolytica, and more preferably from K.pneumoniae or K. oxytoca.

According to another aspect the present invention provides anantagonist, which binds or is capable of binding to an antigen, or anactive fragment or active variant thereof as disclosed in the presentinvention. According to a still further aspect the antagonist accordingto the present invention is an antagonist which is capable of reducingor inhibiting the interaction activity of an antigen, or an activefragment thereof or an active variant thereof, according to the presentinvention to its interaction partner. Such interaction partner is, in apreferred embodiment, an antibody or a receptor, preferably aphysiological receptor, of said antigen, or an active fragment thereofor an active variant thereof.

According to another aspect the present invention provides a method foridentifying an antagonist capable of binding to an antigen, or an activefragment or an active variant thereof, as defined above, comprising:

-   -   a) contacting an isolated or immobilized antigen, or an active        fragment or an active variant thereof, as defined above, with a        candidate antagonist under conditions to permit binding of said        candidate antagonist to said antigen, or an active fragment or        active variant thereof, in the presence of a component capable        of providing a detectable signal in response to the binding of        the candidate antagonist to said antigen, or an active fragment        or an active variant thereof; and    -   b) detecting the presence or absence of a signal generated in        response to the binding of the antagonist to said antigen, or an        active fragment or active variant thereof.

The problem underlying the present invention is further solved by amethod for identifying an antagonist capable of reducing or inhibitingthe interaction activity of an antigen, or an active fragment or anactive variant thereof, as defined above, to its interaction partnercomprising:

-   -   a) providing an antigen, or an active fragment or active variant        thereof, as defined above,    -   b) providing an interaction partner to said antigen, or said        active fragment or active variant thereof, especially an        antibody as defined above,    -   c) allowing interaction of said antigen, or said active fragment        or active variant thereof, to said interaction partner to form        an interaction complex,    -   d) providing a candidate antagonist,    -   e) allowing a competition reaction to occur between the        candidate antagonist and the interaction complex,    -   f) determining whether the candidate antagonist inhibits or        reduces the interaction activities of the antigen, or the active        fragment or the active variant thereof, with the interaction        partner.

The present invention further relates to the use of any of the antigens,or an active fragment or an active variant thereof, as defined above,for the isolation and/or purification and/or identification of aninteraction partner of said antigen, or said active fragment or activevariant thereof.

Another aspect of the present invention relates to a method fordiagnosing an infection with a Klebsiella organism comprising the stepsof:

-   -   a) contacting a sample obtained from a subject with an antigen,        or an active fragment or active variant thereof, as defined        above; and    -   b) detecting the presence of an antibody against said Klebsiella        organism in the sample.

In yet another aspect the present invention provides a method fordiagnosing an infection with a Klebsiella organism comprising the stepsof:

-   -   a) contacting a sample obtained from a subject with the antibody        as defined above; and    -   b) detecting the presence of an antigen of said Klebsiella        organism in the sample.

In an embodiment of said method the antigen of said Klebsiella organismis an antigen, or an active fragment or an active variant thereof, asdefined above.

Still another aspect relates to a method for diagnosing an infectionwith a Klebsiella organism comprising the steps of:

-   -   a) contacting a sample obtained from a subject with a primer or        a probe specific for a nucleic acid molecule, or a fragment        thereof, as defined above; and    -   b) detecting the presence of such nucleic acid molecule or        fragment thereof in the sample.

The present invention also provides a process for in vitro diagnosing adisease related to expression of an antigen or a fragment thereofaccording to the present invention comprising determining the presenceof a nucleic acid sequence encoding said antigen or fragment thereofaccording to the present invention or determining the presence of theantigen or fragment thereof according to the present invention.

In an embodiment of any of the above described methods for diagnosing aninfection with a Klebsiella organism the Klebsiella organism is apathogenic Klebsiella organism, more preferably a Klebsiella organismselected from the group comprising K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca.

Moreover, the present invention provides the use of an antigen, or afragment or a variant thereof, as defined in the present invention forthe generation of a peptide binding to said antigen, or a fragmentthereof or a variant thereof, wherein the peptide is an anticaline.

Moreover, the present invention provides the use of an antigen, or anactive fragment or active variant thereof, as defined above, for thepreparation of a functional nucleic acid, wherein the functional nucleicacid is selected from the group comprising aptamers and spiegelmers.

In another aspect, the present invention provides the use of a nucleicacid molecule as defined above for the preparation of a functionalribonucleic acid, wherein the functional ribonucleic acid is selectedfrom the group comprising ribozymes, antisense nucleic acids and siRNA.

The problem underlying the present invention is further solved by amethod for the treatment of a Klebsiella infection in an animal or humanpreferably in need thereof, comprising the step of administering to saidanimal or human a therapeutically effective amount of an antigen, or anactive fragment or an active variant thereof, or a nucleic acidmolecule, or a vector, or an antibody or a pharmaceutical composition asdefined in any of the preceding aspects.

In an embodiment said Klebsiella infection is an infection with K.pneumoniae including the three subspecies pneumoniae, ozaenae andrhinoscleromatis, K. oxytoca, K. planticola, K. terrigena, and K.ornithinolytica, and more preferably from K. pneumoniae or K. oxytoca.

The problem underlying the present invention is solved in another aspectby a method for immunizing an animal or human against infection with aKlebsiella organism, comprising the step of administering to said animalor human an effective amount of the antigen, or an active fragment or anactive variant thereof, as defined above, or the nucleic acid moleculeas defined above, or a vector as defined above, or an antibody asdefined above, or a pharmaceutical composition as defined above, whereinthe effective amount is suitable to elicit an immune response in saidanimal or human.

In an embodiment of said method for immunizing an animal or humanagainst infection with a Klebsiella organism the Klebsiella organism isselected from the group comprising K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca.

The problem underlying the present invention is solved in yet anotheraspect by a method for stimulating an immune response in an animal orhuman against a Klebsiella organism, comprising the step ofadministering to said animal or human an effective amount of theantigen, or an active fragment or an active variant thereof, as definedabove, or the nucleic acid molecule as defined above or a vector asdefined above, or an antibody as defined above, or a pharmaceuticalcomposition as defined above, wherein the effective amount is suitableto stimulate the immune response in said animal or human.

In an embodiment of said method for stimulating an immune response in ananimal or human against a Klebsiella organism the Klebsiella organism isselected from the group comprising K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca.

It is within the present invention that the various methods and uses,respectively, where an antigen as defined in the present invention isused, can also be performed or practiced using a fragment of suchantigen, preferably an active fragment thereof, or a variant of suchantigen, preferably an active variant thereof, each as preferablydescribed herein. It is also within the present invention that thevarious kinds of compounds disclosed herein as interacting with ortargeting the antigen according to the present invention, canadditionally or alternatively interact with or target the activefragment or the active variant of said antigen.

It is also within the present invention that each and any method in thepractice of which an antibody is used, can, in principle, also bepracticed when instead of the antibody the anticalines or the functionalnucleic acids as defined herein are used, whereby it is preferred thatsuch functional nucleic acid is selected from the group comprisingaptamers and spiegelmers. This applies equally to the various uses ofthe present application.

In a preferred embodiment a fragment of an antigen as disclosed hereinis a part of such antigen which exhibits at least one feature of suchantigen. Preferably such feature is a feature selected from the groupcomprising suitability for the treatment of infections, immunization ofan animal including human, and/or stimulation of an immune response inan animal including human.

It is also within the present invention that any disclosure made hereinin relation to Klebsiella and more specifically K. pneumoniae is equallyapplicable to any Klebsiellae or Klebsiella species, whereby theKlebsiella species is preferably selected from the group comprising K.pneumoniae including the three subspecies pneumoniae, ozaenae andrhinoscleromatis, K. oxytoca, K. planticola, K. terrigena, and K.ornithinolytica, and more preferably from K. pneumoniae or K. oxytoca.

The terms “polypeptide”, “peptide”, “protein” or “antigen” are usedinterchangeably throughout the present specification and refer in acomprehensive manner to the antigen according to the present invention,including each and any variant, fragment, analogue or derivativethereof, particularly as described herein. Insofar, whenever the termpolypeptide, peptide, protein or antigen is used herein, and if notexplicitly stated otherwise, the respective disclosure is also made foror in relation to any antigen according to the present invention,including each and any variant, fragment, analogue or derivativethereof, particularly as described herein. Also it is to be understoodthat any use or aspect described in connection with any of the abovementioned compounds covered by the term polypeptide, peptide, protein orantigen according to the present invention shall be applicable also toeach and any other of the above mentioned compounds covered by the termpolypeptide, peptide, protein or antigen according to the presentinvention.

The present invention advantageously provides an efficient, relevant andcomprehensive set of isolated nucleic acid molecules and antigensencoded by them, including the active fragments and the active variantsthereof, using an antibody preparation from multiple human plasma poolsand surface expression libraries derived from the genome of K.pneumoniae. Thus, the present invention fulfils a widely felt demand forK. pneumoniae antigens, vaccines, diagnostics and products useful inprocedures for preparing antibodies and for identifying compoundseffective against infections caused by pathogenic Klebsiellae, morepreferably K. pneumoniae including the three subspecies pneumoniae,ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena,and K. ornithinolytica, and most preferably from K. pneumoniae or K.oxytoca.

An effective vaccine should be composed of proteins or polypeptides,which are expressed by all strains and are able to induce high affinity,abundant antibodies against cell surface components of said pathogenicKlebsiellae, especially K. pneumoniae including the three subspeciespneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K.terrigena, and K. ornithinolytica, and more preferably from K.pneumoniae or K. oxytoca. The antibodies should be IgG1 and/or IgG3 foropsonization, and any IgG subtype for neutralisation of adherence andtoxin action. A chemically defined vaccine must be definitely superiorcompared to a whole cell vaccine (attenuated or killed), sincecomponents of said pathogenic Klebsiellae, especially K. pneumoniaeincluding the three subspecies pneumoniae, ozaenae and rhinoscleromatis,K. oxytoca, K. planticola, K. terrigena, and K. ornithinolytica, andmore preferably from K. pneumoniae or K. oxytoca, which cross-react withhuman tissues or inhibit opsonization can be eliminated, and theindividual polypeptides inducing protective antibodies and/or aprotective immune response can be selected.

In a preferred embodiment of the present invention, the nucleic acidmolecules exhibit 70% identity over their entire length to a nucleotidesequence set forth in Seq ID Nos 1 to 187 and Seq ID No 375. Morepreferred are nucleic acids that comprise a region that is at least 80%or at least 85% identical over their entire length to a nucleic acidmolecule set forth in Seq ID Nos 1 to 187 and Seq ID No 375. In thisregard, nucleic acid molecules, which are at least 90%, 91%, 92%, 93%,94%, 95%, or 96% identical over their entire length to the same areparticularly preferred. Furthermore, those with at least 97% are highlypreferred, those with at least 98% and at least 99% are particularlyhighly preferred, with at least 99% or 99.5% being the more preferred,with 100% identity being especially preferred. Moreover, preferredembodiments in this respect are nucleic acids, which encode antigens orfragments thereof (polypeptides), which retain substantially the samebiological function or activity as the mature polypeptide set forth inthe Seq ID Nos 188 to 374 and Seq ID No 376. It is also within thepresent invention that the nucleic acid molecules according to thepresent invention are coding for a protein which is preferably anantigen. Still further it is within the present invention, that themolecules defined by Seq ID Nos 188 to 374 and Seq ID No 376 areproteins, which are preferably antigens.

Identity, as known in the art and used herein, is the relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. Identity canbe readily calculated. While there exist a number of methods to measureidentity between two polynucleotide or two polypeptide sequences, theterm is well known to skilled artisans (e.g. Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity are codifiedin computer programs. Preferred computer program methods to determineidentity between two sequences include, but are not limited to, GCGprogram package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA(Altschul, S. et al., 1990).

As a second alternative to the nucleic acid molecules described hereinby reference to Seq ID Nos 1-187 and Seq ID No 375, the description ofwhich is also referred to herein as first alternative, the nucleic acidmolecules according to the present invention can also be nucleic acidmolecules, which are at least essentially complementary to the nucleicacids described in accordance with the first alternative herein. It willbe acknowledged by the ones skilled in the art that an individualnucleic acid molecule is at least essentially complementary to anotherindividual nucleic acid molecule. As used herein complementary meansthat a nucleic acid strand is base pairing via Watson-Crick base pairingwith a second nucleic acid strand. Essentially complementary as usedherein means that the base pairing is not occurring for all of the basesof the respective strands but leaves a certain number or percentage ofthe bases unpaired or wrongly paired. The percentage of correctlypairing bases is preferably at least 70%, more preferably 80%, even morepreferably 90% and most preferably any percentage higher than 90%. Suchhigher percentage includes 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%,whereby such definition is applicable to each aspect of the presentapplication where this kind of terminology is used. It is to be notedthat a percentage of 70% matching bases is considered as homology andthe hybridisation having this extent of matching base pairs isconsidered as stringent. Hybridisation conditions for this kind ofstringent hybridisation may be taken from Current Protocols in MolecularBiology (John Wiley and Sons, Inc., 1987). More particularly, thehybridisation conditions can be as follows:

-   -   Hybridisation performed e.g. in 5×SSPE, 5×Denhardt's reagent,        0.1% SDS, 100 g/mL sheared DNA at 68° C.    -   Moderate stringency wash in 0.2×SSC, 0.1% SDS at 42° C.    -   High stringency wash in 0.1×SSC, 0.1% SDS at 68° C.

Genomic DNA with a GC content of 50% has an approximate T_(M) of 96° C.For 1% mismatch, the T_(M) is reduced by approximately 1° C.

In addition, any of the further hybridisation conditions describedherein are in principle applicable as well.

Of course, all nucleic acid sequence molecules which encode the samepolypeptide molecule as those identified by the present invention areencompassed by any disclosure of a given coding sequence, since thedegeneracy of the genetic code is directly applicable to unambiguouslydetermine all possible nucleic acid molecules which encode a givenpolypeptide molecule, even if the number of such degenerated nucleicacid molecules may be high. This is also applicable for active fragmentsor active variants of a given antigen, as long as the fragments orvariants encode an antigen being suitable to be used such that the sameeffect can be obtained as if the full-length antigen was used.Preferably, such antigens or active fragments or active variants thereofmay be used in a vaccination application, e.g. as an active or passivevaccine.

As a third alternative, the nucleic acid molecule according to thepresent invention can also be a nucleic acid which comprises a stretchof at least 15 bases of the nucleic acid molecule according to the firstor second alternative of the nucleic acid molecules according to thepresent invention as outlined above. Preferably, the bases form acontiguous stretch of bases. However, it is also within the scope of thepresent invention that the stretch consists of two or more moieties,which are separated by a number of bases.

The nucleic acid molecules according to the present invention maypreferably consist of at least 20, even more preferred at least 30,especially at least 50 contiguous bases from the sequences disclosedherein. The suitable length may easily be optimised due to the intendedfield of use (e.g. as (PCR) primers, probes, capture molecules (e.g. ona (DNA) chip), etc.). Preferred nucleic acid molecules contain at leasta contiguous 15 base portion of one or more of the immunogenic aminoacid sequences listed in Tables 1 and 4. Specifically preferred arenucleic acids containing a contiguous portion of a DNA sequence of anysequence contained in the sequence protocol of the present applicationwhich shows 1 or more, preferably more than 2, especially more than 5,non-identical nucleic acid residues compared to the unfinished genomesequences of K. pneumoniae MGH78578 and Kp342 that are available(http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi, orhttp://pedant.gsf.de/, and http://cmr.tigr.org/) and plasmids asspecified in the following by their accession numbers K. pneumoniaeplasmid pJHCMW1, NC_(—)003486, K. pneumoniae plasmid pIP843,NC_(—)005015, K. pneumoniae plasmid pK2044, NC_(—)006625, K. pneumoniaeplasmid pKlebB-k17-80, NC_(—)002610, K. pneumoniae plasmid pKPN2,NC_(—)005018, K. pneumoniae plasmid pLVPK, NC_(—)005249, Klebsiella sp.KCL-2 plasmid pMGD2, NC_(—)003789. However, the unfinished genomesequences mentioned above are subject to continuous resequencing,corrections, amendments and additions. For example, as of April 2007 thesequence available at http://cmr.tigr.org/ is in status 7 out of 9status codes, which means that additional sequences generated for gapclosure have been assembled and added to data release(http://www.tigr.org/tdb/mdb/mdbinprogress.html). Specifically preferrednon-identical nucleic acid residues are residues, which lead to anon-identical amino acid residue. Preferably, the nucleic acid sequencesencode polypeptides, proteins, or antigens having at least 1, preferablyat least 2, preferably at least 3 different amino acid residues comparedto the published or listed K. pneumoniae counterparts mentioned above.Preferably, this kind of polypeptides, proteins, or antigens still hasat least one of the characteristics of the molecules disclosed hereinhaving identical amino acid residues. Also preferred are such isolatedpolypeptides, which are fragments of the proteins or of the antigensdisclosed herein, e.g. in the Sequence Listing, having at least 6, 7, or8 amino acid residues and being encoded by the nucleic acids asdescribed herein.

The nucleic acid molecule according to the present invention can, as afourth alternative, also be a nucleic acid molecule which anneals understringent hybridisation conditions to any of the nucleic acids of thepresent invention according to the first, second, or third alternativeas disclosed herein. Stringent hybridisation conditions are typicallythose described herein.

Finally, the nucleic acid molecule according to the present inventioncan, as a fifth alternative, also be a nucleic acid molecule which, butfor the degeneracy of the genetic code, would hybridise to any of thenucleic acid molecules of the present invention according to the first,second, third, and fourth alternative as outlined herein. This kind ofnucleic acid molecule refers to the fact that preferably the nucleicacids according to the present invention code for the antigen, orfragments or variants thereof, according to the present invention. Thiskind of nucleic acid molecule is particularly useful in the detection ofa nucleic acid molecule according to the present invention and thus thediagnosis of the respective microorganisms such as K. pneumoniae or anypathogenic Klebsiellae, particularly those pathogenic Klebsiella speciesdisclosed herein, and any disease or diseased condition where thesekinds of microorganism are involved. Preferably, such microorganism,especially an opportunistic microorganism, is causing such diseasedirectly or indirectly. Preferably, the hybridisation could occur or bepreformed under stringent conditions as described in connection with thefourth alternative described herein.

Nucleic acid molecule as used herein generally refers to any ribonucleicacid molecule or deoxyribonucleic acid molecule, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, nucleic acidmolecule as used herein refers to, among others, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded RNA, and RNA that is a mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded, or a mixture of single- and double-stranded regions. Inaddition, nucleic acid molecule as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may be derived from one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide. Asused herein, the term nucleic acid molecule includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“nucleic acid molecule” as that term is intended herein. Moreover, DNAsor RNAs comprising unusual bases, such as inosine, or modified bases,such as tritylated bases, to name just two examples, are nucleic acidmolecule as the term is used herein. It will be appreciated that a greatvariety of modifications can be made to DNA and RNA that serve manyuseful purposes known to those of skill in the art. The term nucleicacid molecule as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of nucleic acid molecule,as well as the chemical forms of DNA and RNA characteristic of virusesand cells, including simple and complex cells, inter alia. The termnucleic acid molecule also embraces short nucleic acid molecules oftenreferred to as oligonucleotide(s). “Polynucleotide” and “nucleic acid”or “nucleic acid molecule” are often used interchangeably herein.

Nucleic acid molecules provided in the present invention also encompassnumerous unique fragments, both longer and shorter than the nucleic acidmolecule sequences set forth in the sequencing listing of the presentapplication, more specifically of the K. pneumoniae coding regions,which can be generated by standard cloning methods. To be unique, afragment must be of sufficient size to distinguish it from other knownnucleic acid sequences, most readily determined by comparing anyselected K. pneumoniae fragment to the nucleotide sequences inbiosequence databases such as GenBank. It will be appreciated by the oneskilled in the art that what is said herein in any aspect in relation toK. pneumoniae applies equally to any of the other Klebsiella speciesdescribed herein, more preferably any pathogenic Klebsiella speciesdescribed herein.

Additionally, modifications can be made to the nucleic acid moleculesand polypeptides that are encompassed by the present invention. Forexample, the nucleic acid also includes sequences that are a result ofthe degeneration of the genetic code. There are 20 natural amino acids,most of which are specified by more than one codon. Thus, nucleotidesubstitutions can be made which do not affect the polypeptide encoded bythe nucleic acid. Accordingly, any nucleic acid molecule which encodesan antigen or fragments thereof is encompassed by the present invention.

Furthermore, any of the nucleic acid molecules encoding antigens orfragments thereof provided by the present invention can be functionallylinked, using standard techniques such as standard cloning techniques,to any desired regulatory sequences, whether an K. pneumoniae regulatorysequence or a heterologous regulatory sequence, heterologous leadersequence, heterologous marker sequence or a heterologous coding sequenceto create a fusion protein.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA or cRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or produced bychemical synthetic techniques or by a combination thereof. The DNA maybe triple-stranded, double-stranded or single-stranded. Single-strandedDNA may be the coding strand, also known as the sense strand, or it maybe the non-coding strand, also referred to as the anti-sense strand.

The present invention further relates to variants of the nucleic acidmolecules described herein which encode fragments, analogs andderivatives of the antigens and fragments thereof having a deducted K.pneumoniae amino acid sequence set forth in the Sequence Listing. Avariant of the nucleic acid molecule may be a naturally occurringvariant such as a naturally occurring allelic variant, or it may be avariant that is not known to occur naturally. Such non-naturallyoccurring variants of the nucleic acid molecule may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells or organisms.

Among variants in this regard are variants that differ from theaforementioned nucleic acid molecules by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or additions. Preferred are nucleic acid molecules encoding avariant, analog, derivative or fragment, or a variant, analogue orderivative of a fragment, which have an K. pneumoniae sequence as setforth in the Sequence Listing, in which several, a few, 5 to 10, 1 to 5,1 to 3, 2, 1 or no amino acid(s) is substituted, deleted or added, inany combination. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the K. pneumoniae polypeptides set forth inthe Sequence Listing. Also especially preferred in this regard areconservative substitutions.

The nucleic acid molecules of the present invention may also be used asa hybridisation probe for, e.g., RNA, cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding polypeptides of thepresent invention and to isolate cDNA and genomic clones of other genesthat have a high sequence similarity to the nucleic acid molecules ofthe present invention. Such probes generally will comprise at least 15bases. Preferably, such probes will have at least 20, at least 25 or atleast 30 bases, and may have at least 50 bases. Particularly preferredprobes will have at least 30 bases, and will have 50 bases or less, suchas 30, 35, 40, 45, or 50 bases.

For example, the coding region of a nucleic acid molecule of the presentinvention may be isolated by screening a relevant library using theknown DNA sequence to synthesize an oligonucleotide probe. A labelledoligonucleotide having a sequence complementary to that of a gene of thepresent invention is then used to screen a library of cDNA, genomic DNAor mRNA to determine to which members of the library the probehybridizes.

The nucleic acid molecules and polypeptides of the present invention maybe employed as reagents and materials for the development or preparationof pharmaceutical compositions and/or diagnostics for diseases,particularly human disease, as further discussed herein.

The nucleic acid molecules of the present invention that areoligonucleotides can be used in the processes herein as described, butpreferably for PCR, to determine whether or not the K. pneumoniae genesidentified herein in whole or in part are present and/or transcribed ininfected tissue such as skin, synovia or blood. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained. For this andother purposes arrays which are known as such in the art, comprising atleast one of the nucleic acids or polypeptides according to the presentinvention as described herein, may be used.

The nucleic acid molecules according to the present invention may beused for the detection of nucleic acid molecules and organisms orsamples containing these nucleic acids. Preferably such detection is fordiagnosis, more preferably for the diagnosis of a disease related orlinked to the presence or abundance of Klebsiellae or any other pathogenspecies of Klebsiella, especially K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca.

Eukaryotes (herein also “individual(s)”), particularly mammals, andespecially humans, infected with Klebsiellae or any other pathogenspecies of Klebsiella, especially K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca can be identified by detecting any ofthe nucleic acid molecules according to the present invention detectedat the DNA level by a variety of techniques. Preferred nucleic acidmolecule candidates for distinguishing Klebsiellae or said otherpathogenic Klebsiella from other organisms can be obtained.

The invention provides a process for diagnosing disease, arising frominfection with Klebsiellae or any other pathogen species of Klebsiella,especially K. pneumoniae including the three subspecies pneumoniae,ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena,and K. ornithinolytica, and more preferably from K. pneumoniae or K.oxytoca, comprising determining from a sample isolated or derived froman individual an increased level of expression of a nucleic acidmolecule having the sequence of a nucleic acid molecule as disclosedherein and more preferably set forth in the Sequence Listing. Expressionof nucleic acid molecules can be measured using any one of the methodswell known in the art for the quantification of nucleic acid molecules,such as, for example, PCR, RT-PCR, RNase protection, Northern blotting,other hybridisation methods and the arrays described herein.

Isolated as used herein means separated “by the hand of man” from itsnatural state; i.e., that, if it occurs in nature, it has been changedor removed from its original environment, or both. For example, anaturally occurring nucleic acid molecule or a polypeptide naturallypresent in a living organism in its natural state is not “isolated”, butthe same nucleic acid molecule or polypeptide separated from thecoexisting materials of its natural state is “isolated”, as the term isemployed herein. As part of or following isolation, such nucleic acidmolecules can be joined to other nucleic acid molecules, such as DNAs,for mutagenesis, to form fusion proteins, and for propagation orexpression in a host, for instance. The isolated nucleic acid molecules,alone or joined to other nucleic acid molecules such as vectors, can beintroduced into host cells, in culture or in whole organisms. Introducedinto host cells in culture or in whole organisms, such DNAs still wouldbe isolated, as the term is used herein, because they would not be intheir naturally occurring form or environment. Similarly, the nucleicacid molecules and polypeptides may occur in a composition, such as amedia formulations, solutions for introduction of nucleic acid moleculesor polypeptides, for example, into cells, compositions or solutions forchemical or enzymatic reactions, for instance, which are not naturallyoccurring compositions, and, therein remain isolated nucleic acidmolecules or polypeptides within the meaning of that term as it isemployed herein.

The nucleic acid molecules of the present invention may be originallyformed in vitro, e.g. by chemical synthesis, or in a cell culture andsubsequent isolation or purification. In general, the nucleic acids maybe obtained by the manipulation of nucleic acids by endonucleases and/orexonucleases and/or polymerases and/or ligases and/or recombinases orother methods known to the skilled practitioner to produce the nucleicacids.

The nucleic acid sequences as defined by Seq ID Nos 1 to 187 and Seq IDNo 375 start with the first complete codon comprised by the fragment asinserted into the vector and encodes the first amino acid as defined bySeq ID Nos 188 to 374 and Seq ID No 376. However, for the recombinantproduction additional nucleic acids might be useful or necessary tofacilitate the cloning and expression.

Preferably, the nucleic acids can be isolated from Klebsiellae or anyother pathogen species of Klebsiella, especially K. pneumoniae includingthe three subspecies pneumoniae, ozaenae and rhinoscleromatis, K.oxytoca, K. planticola, K. terrigena, and K. ornithinolytica, and morepreferably from K. pneumoniae or K. oxytoca by methods known to the oneskilled in the art. The same applies to the polypeptides according tothe present invention.

The present invention also relates to vectors, which comprise a nucleicacid molecule or nucleic acid molecules of the present invention. Avector may additionally include nucleic acid sequences that permit it toreplicate in the host cell, such as an origin of replication, one ormore therapeutic genes and/or selectable marker genes and other geneticelements known in the art such as regulatory elements directingtranscription, translation and/or secretion of the encoded protein. Thevector may be used to transduce, transform or infect a cell, therebycausing the cell to express nucleic acids and/or proteins other thanthose native to the cell. The vector optionally includes materials toaid in achieving entry of the nucleic acid into the cell, such as aviral particle, liposome, protein coating or the like.

The present invention also relates to host cells, which are geneticallyengineered with vectors of the invention and to the production of thepolypeptides according to the present invention by recombinanttechniques.

A great variety of expression vectors can be used to express thepolypeptides according to the present invention. Generally, any vectorsuitable to maintain, propagate or express nucleic acids to express apolypeptide in a host may be used for expression in this regard. Inaccordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Starting plasmidsdisclosed herein are either commercially available, publicly available,or can be constructed from available plasmids by routine application ofwell-known, published procedures. Preferred among vectors, in certainrespects, are those for expression of nucleic acid molecules and thepolypeptides according to the present invention. Nucleic acid constructsin host cells can be used in a conventional manner to produce the geneproduct encoded by the recombinant sequence. Alternatively, thepolypeptides according to the preset invention can be syntheticallyproduced by conventional peptide synthesizers. Mature proteins can beexpressed in mammalian cells, yeast, bacteria, or other cells under thecontrol of appropriate promoters. Cell-free translation systems can alsobe employed to produce such proteins using RNAs derived from the DNAconstruct of the present invention.

Host cells can be genetically engineered to incorporate nucleic acidmolecules and express nucleic acid molecules of the present invention.Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanomacells; and plant cells.

The host cells can be transfected, e.g. by conventional means such aselectroporation with at least one expression vector containing a nucleicacid of the invention under the control of a transcriptional regulatorysequence.

According to another aspect of the present invention, a comprehensiveset of novel polypeptides is provided. Such polypeptides, as mentionedpreviously herein, are antigens as disclosed herein, and the fragmentsthereof, preferably the active fragments thereof, and the variantsthereof, preferably the active variants thereof. Preferably, thepolypeptides according to the present invention are antigens andfragments thereof. In a preferred embodiment of the invention, anantigen comprising an amino acid sequence being preferably encoded byany one of the nucleic acids molecules and fragments thereof asdescribed herein, are provided. In another preferred embodiment of theinvention a novel set of proteins and antigens and active fragments aswell as active variants thereof is provided which comprise amino acidsequences selected from the group comprising Seq ID Nos 188 to 374 andSeq ID No 376.

The polypeptides according to the present invention, i.e. the antigens,as provided by the present invention preferably include any polypeptideor molecule set forth in the Sequence Listing as well as polypeptideswhich have at least 70% identity to such polypeptide according to thepresent invention, preferably at least 80% or 85% identity to suchpolypeptide according to the present invention, and more preferably atleast 90% similarity (more preferably at least 90% identity) to suchpolypeptide according to the present invention and more preferably asset forth in the Sequence Listing and still more preferably at least95%, 96%, 97%, 98%, 99% or 99.5% similarity (still more preferably atleast 95%, 96%, 97%, 98%, 99%, or 99.5% identity) to such polypeptideaccording to the present invention and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 4 amino acids and more preferably at least 8, still morepreferably at least 30, still more preferably at most 50 amino acids,such as 4, 8, 10, 20, 30, 35, 40, 45 or 50 amino acids. In a preferredembodiment such portions are active fragments of the polypeptidesaccording to the present invention.

The invention also relates to fragments, analogs, and derivatives of thepolypeptides according to the present invention. The terms “fragment”,“derivative” and “analog” when referring to such polypeptide whose aminoacid sequence is preferably set forth in the Sequence Listing, means apolypeptide which retains essentially the same or a similar biologicalactivity as such polypeptide. It will be acknowledged by the onesskilled in the art that the meaning of the term “similar biologicalactivity” as used herein preferably depends on the polypeptide underconsideration and more specifically its function. The term “biologicalactivity” as used herein is further defined below. More preferably, asimilar biological function or activity differs from the function of thenon-fragment or the non-derivative in terms of extent of activity,affinity, immunogenicity, stability and/or specificity. In a preferredembodiment the difference is less than 50%, less than 75% or less than90%.

In an embodiment the fragment, derivative, variant or analog of apolypeptide according to the present invention is 1) one in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or 2) one in which one or more of the aminoacid residues includes a substituent group, or 3) one in the polypeptideaccording to the present invention or a fragment thereof is fused withanother compound, such as a compound to increase the half-life of thepolypeptide according to the present invention or a fragment thereofsuch as, for example, polyethylene glycol, or 4) one in which theadditional amino acids are fused to the polypeptide according to thepresent invention or a fragment thereof, such as a leader or secretorysequence or a sequence which is employed for purification of saidpolypeptide according to the present invention or fragment thereof or aproprotein sequence. Such fragments, derivatives, variants and analogsare deemed to be within the scope of those skilled in the art from theteachings herein.

The present invention also relates to proteins and antigens of differentKlebsiella species, preferably pathogenic Klebsiella species, especiallyK. pneumoniae including the three subspecies pneumoniae, ozaenae andrhinoscleromatis, K. oxytoca, K. planticola, K. terrigena, and K.ornithinolytica, and more preferably from K. pneumoniae or K. oxytocawhich are preferably homologues. Such homologues may easily be isolatedbased on the nucleic acid and amino acid sequences disclosed herein.There are multiple serotypes or clinical strains distinguished to datefor each of the pathogens and the typing is based on serotype specificantisera or molecular approaches. The presence of any antigen canaccordingly be determined for every serotype. The contribution of thevarious serotypes to the different Klebsiella infections varies indifferent age groups and especially geographical regions. Particularlyrelevant serotypes of Klebsiella are, for example, K1, K2, K3, K10, K21,K22, K30, K55, K64, O1, O2a, O3, O4, O5, or O12, or any combination ofsaid K and said O serotypes. It is an important aspect that the mostvaluable protective antigens need to be conserved among various clinicalstrains.

Additionally, fusion polypeptides comprising such antigens, variants,analogs, derivatives and fragments thereof, and variants, analogs andderivatives of the fragments are also encompassed by the presentinvention. Such fusion polypeptides and proteins, as well as nucleicacid molecules encoding them, can readily be made using standardtechniques, including standard recombinant techniques for producing andexpression of a recombinant polynucleic acid encoding a fusion protein.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide according to the presentinvention by another amino acid of like characteristics. Typically seenas conservative substitutions are the replacements, one for another,among the aliphatic amino acids Ala, Val, Leu and Ile; interchange ofthe hydroxyl residues Ser and Thr, exchange of the acidic residues Aspand Glu, substitution between the amide residues Asn and Gln, exchangeof the basic residues Lys and Arg and replacements among the aromaticresidues Phe and Tyr.

In another embodiment of the invention the peptide as defined above maybe modified by a variety of chemical techniques to produce derivativeshaving essentially the same activity (as defined above for fragments andvariants) as the modified peptides, and optionally having otherdesirable properties. For example, carboxylic acid groups of theprotein, whether C-terminal or side chain, may be provided in the formof a salt of a pharmaceutically acceptable cation or esterified to forman ester, or converted to an amide. Amino groups of the peptide, whetheramino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be converted to an amide. Hydroxyl groups of the peptideside chains may be converted to alkoxy or to an ester using wellrecognized techniques. Phenyl and phenolic rings of the peptide sidechains may be substituted with one or more halogen atoms, such asfluorine, chlorine, bromine or iodine, or with alkyl, alkoxy, carboxylicacids and esters thereof, or amides of such carboxylic acids. Thiols canbe protected with any one of a number of well recognized protectinggroups, such as acetamide groups.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of any polypeptide accordingto the present invention as disclosed herein and preferably set forth inthe Sequence Listing, in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the peptide of the present invention. Also especiallypreferred in this regard are conservative substitutions. Most highlypreferred are peptides having an amino acid sequence set forth in theSequence Listing without substitutions.

Variants of any of the antigens in their various embodiments disclosedherein and in particular the antigens and peptides specified herein bySeq ID Nos 188 to 374 and Seq ID No 376, can typically also becharacterized by means of bioinformatics. Respective tools such as theNCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990)are available from several sources, including the National Center forBiotechnology Information (NCBI, Bethesda, Md.) and on the Internet, foruse in connection with the sequence analysis programs blastp, blastn,blastx, tblastn and tblastx. Using the NCBI Blast 2.0, gapped blastp setto default parameters. For comparisons of amino acid sequences of atleast 35 amino acids, the Blast 2 sequences function is employed usingthe default BLOSUM62 matrix set to default parameters, (gap existencecost of 11, and a per residue gap cost of 1). When aligning shortpeptides (fewer than around 35 amino acids), the alignment is performedusing the Blast 2 sequences function, employing the PAM30 matrix set todefault parameters (open gap 9, extension gap 1 penalties). Methods fordetermining sequence identity over such short windows such as 15 aminoacids or less are described at the website that is maintained by theNational Center for Biotechnology Information in Bethesda, Md.(http://www.ncbi.nlm.nih.gov/BLAST/).

The active variant of an antigen is obtained by sequence alterations inthe antigen, including each and any variant, fragment, analogue orderivative thereof, if not explicitly indicated to the contrary, whereinthe polypeptide according to the present invention with the sequencealterations retains a function of the unaltered polypeptide according tothe present invention, e.g. having a biological activity similar to thatdisplayed by the complete antigen, including the ability to induce animmune response and/or to show protection against a Klebsiella organisme.g. in a sepsis and/or lethality model. A further example of retainingthe function of the unaltered polypeptide according to the presentinvention is that the active variant of the antigen specifically binds apolypeptide specific antibody that binds an unaltered form of thepolypeptide according to the present invention. By “biological function”or “biological activity” is preferably meant a function of thepolypeptide in cells or organisms in which it naturally occurs, even ifthe function is not necessary for the growth or survival of the cellsand organisms, respectively. For example, the biological function of aporin is to allow the entry into cell of compounds present in theextracellular medium. The biological function is distinct from theantigenic function. A polypeptide according to the present invention canhave more than one biological function.

The sequence alterations of such variants can include, but are notlimited to, conservative substitutions, deletions, mutations andinsertions. Preferably, the active variant exhibits reactivity withhuman sera of septicaemia patients, more preferably mediatesseroconversion and most preferably shows bactericidal activity. Thesecharacteristics of the active variant can be assessed e.g. as detailedin the Examples. In the context of the present invention a variantspecifically binds a specific antibody (preferably being polyclonalantibodies raised against recombinant proteins in animals such as mouse,rabbit or monoclonal antibodies generated in mouse), exhibits reactivitywith human sera from patients with septicaemia, mediates seroconversionor shows bactericidal activity, if the activity of the variant amountsto at least 10%, preferably at least 25%, more preferably at least 50%,even more preferably at least 70%, still more preferably at least 80%,especially at least 90%, particularly at least 95%, most preferably atleast 99% of the activity of the antigen without sequence alterations.

Said active variants include naturally-occurring allelic variants, aswell as mutants or any other non-naturally occurring variants. As isknown in the art, an allelic variant is an alternate form of a(poly)peptide that is characterized as having a substitution, deletion,or addition of one or more amino acids that does essentially not alterthe biological function of the polypeptide, as it is described above.

Within any species of the living world, allelic variation is the rule.For example, any bacterial species, e.g. K. pneumoniae, is usuallyrepresented by a variety of strains (characterized by clonalreproduction) that differ from each other by minor allelic variations.Indeed, a polypeptide that fulfils the same biological function indifferent strains can have an amino acid sequence that is not identicalin each of the strains. Such an allelic variation is equally reflectedat the polynucleotide level.

Allelic variation is very common within the Klebsiella species asdescribed for class A beta-lactamase K2 (Haeggman, S. et al., 1997).

In a preferred embodiment, the active variant of or the active fragmentderived from the polypeptide according to the present invention by aminoacid exchanges, deletions or insertions may also conserve, or morepreferably improve, the activity (reactivity, seroconversion and/orbactericidal activity as defined herein). Furthermore, thesepolypeptides may also cover epitopes, which trigger the same orpreferably an improved T cell response. These epitopes are referred toas “heteroclitic” as further defined herein. They have a similar orpreferably greater affinity to MHC/HLA molecules, and the ability tostimulate the T cell receptors (TCR) directed to the original epitope ina similar or preferably stronger manner. Heteroclitic epitopes can beobtained by rational design i.e. taking into account the contribution ofindividual residues to binding to MHC/HLA as for instance described byRammensee et al. (1999), combined with a systematic exchange of residuespotentially interacting with the TCR and testing the resulting sequenceswith T cells directed against the original epitope. Such a design ispossible for a skilled person in the art without undue experimentation.

In a still more preferred embodiment of the invention the active variantof a polypeptide according to the present invention is any of thepolypeptides disclosed herein and more specifically any of thepolypeptides defined by the Seq ID Nos 188 to 374 and Seq ID No 376,having at least 50% sequence identity to the polypeptides of any of saidSeq ID Nos 188 to 374 and Seq ID No 376, especially at least 60%,preferably at least 70%, more preferably at least 80%, still morepreferably at least 90%, even more preferably at least 95%, 96%, 97%,98%, most preferably 99% sequence identity to the polypeptides of any ofsaid Seq ID Nos 188 to 374 and Seq ID No 376 and/or is derived from saidpolypeptides of any of the sequences of Seq ID Nos 188 to 374 and Seq IDNo 376 by conservative substitutions. Conservative substitutions arethose that take place within a family of amino acids that are related intheir side chains and chemical properties. Examples of such families areamino acids with basic side chains, with acidic side chains, withnon-polar aliphatic side chains, with non-polar aromatic side chains,with uncharged polar side chains, with small side chains, with largeside chains etc. In one embodiment, one conservative substitution isincluded in the peptide. In another embodiment, two conservativesubstitutions or less are included in the peptide. In a furtherembodiment, three conservative substitutions or less are included in thepeptide.

Examples of conservative amino acid substitutions include, but are notlimited to, those listed below:

Original Conservative Residue Substitutions Ala Ser Arg Lys Asn Gln; HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu; Val Leu Ile; ValLys Arg; Gln; Asn Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

The polypeptides according to the present invention, and fragments andvariants thereof, also include or consist of modified epitopes whereinpreferably one or two of the amino acids of a given epitope are modifiedor replaced according to the rules disclosed in, e.g., Tourdot, S. etal., 2000, as well as the nucleic acid sequences encoding such modifiedepitopes. The epitopes as presented by the polypeptides according to thepresent invention are also referred to herein as the present epitopes.

It is clear that also epitopes derived from the present epitopes byamino acid exchanges improving, conserving or at least not significantlyimpeding the T cell activating capability of the epitopes are covered bythe epitopes according to the present invention. Therefore the presentepitopes also cover epitopes, which do not contain the original sequenceas derived from K. pneumoniae, but trigger the same or preferably animproved T cell response. These epitope are referred to as“heteroclitic”; they need to have a similar or preferably greateraffinity to MHC/HLA molecules, and the need the ability to stimulate theT cell receptors (TCR) directed to the original epitope in a similar orpreferably stronger manner.

Another possibility for identifying epitopes and more specificallyheteroclitic epitopes includes the screening of peptide libraries with Tcells directed against one or several of the present epitopes. Apreferred way is the positional scanning of synthetic peptide libraries.Such approaches have been described in detail for instance by Hemmer, B.et al., (1999) and the references given therein.

As an alternative to epitopes represented by the present derived aminoacid sequences or heteroclitic epitopes as disclosed herein, alsosubstances or compounds mimicking these epitopes which are also referredto herein as “peptidemimetica” or “retro-inverse-peptides” can beapplied and are thus within the present invention.

Another aspect of the design of improved epitopes is their formulationor modification with substances increasing their capacity to stimulate Tcells. These include T helper cell epitopes, lipids or liposomes orpreferred modifications as described in WO 01/78767.

Another way to increase the T cell stimulating capacity of epitopes istheir formulation with immune stimulating substances for instancecytokines or chemokines like interleukin-2, -7, -12, -18, class I and IIinterferons (IFN), especially IFN-gamma, GM-CSF, TNF-alpha, flt3-ligandand others.

The polypeptides according to the present invention are preferablyprovided in an isolated form, and preferably are purified tohomogeneity.

In another embodiment of the present invention the variant is afragment. The fragment is characterized by being derived from theantigen as defined above by one or more amino acid deletions. Thedeletion(s) may be C-terminally, N-terminally and/or internally.Preferably the fragment is obtained by at most 10, 20, 30, 40, 50, 60,80, 100, 150 or 200, more preferably by at most 10, 20, 30, 40 or 50,even more preferably at most 5, 10 or 15, still more preferably at most5 or 10, most preferably 1, 2, 3, 4 or 5 deletion(s). The activefragment of the invention is characterized by having a biologicalactivity similar to that displayed by the complete antigen, includingthe ability to induce immunization and/or to show protection againstKlebsiella e.g. in a sepsis and/or lethality model. The fragment of anantigen is active in the context of the present invention, if theactivity of the fragment amounts to at least 10%, preferably at least25%, more preferably at least 50%, even more preferably at least 70%,still more preferably at least 80%, especially at least 90%,particularly at least 95%, most preferably at least 99% of the activityof the antigen without sequence alteration. These fragments may bedesigned or obtained in any desired length, including as small as about50 to 80 amino acids in length.

In a further embodiment a fragment, and more preferably a fragment, ofthe polypeptide according to the present invention are characterised bystructural or functional attributes, i.e. fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet and beta-sheetforming regions, turn and turn-forming regions, coil and coil-formingregions, hydrophilic regions, hydrophobic regions, alpha-amphipathicregions, beta-amphipathic regions, flexible regions, surface-formingregions, substrate binding regions, and high antigenic index regions ofthe polypeptide according to the present invention, and combinations ofsuch fragments. Preferred regions are those that mediate antigenicityand antibody binding activities of the polypeptides according to thepresent invention. Most highly preferred in this regard are fragmentsthat have a chemical, biological or other activity of the antigen andfragments thereof of the present invention, including those with asimilar activity or an improved activity, whereby such improvedactivities are immunogenicity and stability, or with a decreasedundesirable activity, whereby such decreased undesirable activity isenzymatic and toxic function and generation of human cross-reactiveantibodies. Particularly preferred are fragments comprising receptors ordomains of enzymes that confer a function essential for viability ofKlebsiellae or any other pathogenic Klebsiella species, or the abilityto cause disease in humans. Further preferred fragments of thepolypeptides according to the present invention are those that compriseor contain antigenic or immunogenic determinants in an animal,especially in a human. Such fragments are also referred to as antigenicfragment.

An antigenic fragment is preferably defined as a fragment, which isantigenic by itself or may be made antigenic when provided as a hapten.Therefore, also antigens or antigenic fragments showing one or,particularly for longer fragments, only a few amino acid exchanges areenabled by the present invention, provided that the antigenicity orantigenic capacities of such fragments with amino acid exchanges are notseverely deteriorated on the exchange(s), i.e., suited for eliciting anappropriate immune response in an individual vaccinated with thisantigen and identified by individual antibody preparations fromindividual sera.

Preferred examples of such fragments of the polypeptides according tothe invention are those listed in Table 16 (SED ID NOs 188-203 and 376).

Further preferred examples of such fragments of the polypeptidesaccording to the present invention are the core amino acid sequence asindicated in column “Predicted immunogenic aa” or “Location ofidentified immunogenic region” of Table 1, or as defined by columns“From aa” and “To aa” of Table 4, or as indicated in column “Location inprotein (aa)” of Table 5.

All these fragments listed in tables 1, 4 and 5 individually and eachindependently form a preferred selected aspect of the present invention.

It will be appreciated that the invention also relates to, among others,nucleic acid molecules encoding the aforementioned fragments, variants,active variants, and active fragments, nucleic acid molecules thathybridise to nucleic acid molecules encoding the fragments, variants,active variants, and active fragments, particularly those that hybridiseunder stringent conditions, and nucleic acid molecules, such as PCRprimers, for amplifying nucleic acid molecules that encode thefragments. In these regards, preferred nucleic acid molecules are thosethat correspond to the preferred fragments, as discussed above.

The polypeptides according to the present invention may be expressed ina modified form, such as a fusion protein, and may include not onlysecretion signals but also additional heterologous functional regions.Thus, for instance, a region of additional amino acids, particularlycharged amino acids, may be added to the N- or C-terminus of thepolypeptide to improve stability and persistence in the host cell,during purification or during subsequent handling and storage. Also,regions may be added to the polypeptide to facilitate purification or toenhance expression. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stability,to enhance expression or to facilitate purification, among others, arefamiliar and routine techniques in the art. A preferred fusion proteincomprises a heterologous region from immunoglobulin that is useful tosolubilize or purify polypeptides. For example, EP 0 464 533 disclosesfusion proteins comprising various portions of constant region ofimmunoglobin molecules together with another protein or part thereof. Indrug discovery, for example, proteins have been fused with antibody Fcportions for the purpose of high-throughout screening assays to identifyantagonists. See for example, (Bennett, D. et al., 1995) and (Johanson,K. et al., 1995). Fusions also may include the polypeptides according tothe present invention fused or coupled to moieties other than aminoacids, including lipids and carbohydrates. Further, antigens of thisinvention may be employed in combination with other vaccinal agentsdescribed by the prior art, as well as with other species of vaccinalagents derived from other microorganisms. Such proteins are useful inthe prevention, treatment and diagnosis of diseases caused by a widespectrum of Klebsiella isolates.

In a further embodiment the peptide of the invention is fused to anepitope tag which provides an epitope to which an anti-tag substance canselectively bind. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the peptide but may be incorporated as an internalinsertion or substitution as the biological activity permits. Thepresence of such epitope-tagged forms of a peptide can be detected usinga substance such as an antibody against the tagged peptide. Also,provision of the epitope tag enables the peptide to be readily purifiedby affinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. Various tag polypeptidesand their respective antibodies are well known in the art. Examplesinclude poly-histidine (poly-his), poly-histidine-glycine (poly-his-gly)tags, the HA tag polypeptide, the c-myc tag, the Strep tag and the FLAGtag.

The polypeptides of the invention may be prepared by any of a number ofconventional techniques. For example, they can be produced by chemicalsynthesis as well as by biotechnological means. The latter comprise thetransfection or transformation of a host cell with a vector containing anucleic acid according to the present invention. In a preferredembodiment the vector is a vector according to the present invention.The biotechnological production of the polypeptides according to thepresent invention further comprises the cultivation of the transfectedor transformed host cell under conditions, that allow expression of theprotein and which are known to the one skilled in the art. The expressedprotein is recovered, isolated, and optionally purified from the cell(or from the culture medium, if expressed extracellularly) byappropriate means known to one of skill in the art. For example, theproteins are isolated in soluble form following cell lysis, or extractedusing known techniques, e.g. in guanidine chloride. The moleculescomprising the polypeptides and antigens of this invention may befurther purified using any of a variety of conventional methodsincluding, but not limited to: ammonium sulfate or ethanolprecipitation, acid extraction, liquid chromatography such as normal orreversed phase, using HPLC, FPLC and the like; affinity chromatography(such as with inorganic ligands or monoclonal antibodies), sizeexclusion chromatography, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography, lectin chromatography, immobilized metalchelate chromatography, gel electrophoresis, and the like. One of skillin the art may select the most appropriate isolation and purificationtechniques without departing from the scope of this invention. Suchpurification provides the antigen in a form substantially free fromother proteinaceous and non-proteinaceous materials of themicroorganism.

An alternative approach to prepare polypeptides according to theinvention involves generating the fragments of known peptides byenzymatic digestion, e.g., by treating the protein with an enzyme knownto cleave proteins at sites defined by particular amino acid residues,or by digesting the DNA with suitable restriction enzymes, expressingthe digested DNA and isolating the desired fragment. Yet anothersuitable technique involves isolating and amplifying a DNA fragmentencoding a desired peptide fragment, by polymerase chain reaction (PCR).Oligonucleotides that define the desired termini of the DNA fragment areemployed as the 5′ and 3′ primers in the PCR. Techniques for makingmutations, such as deletions, insertions and substitutions, atpredetermined sites in DNA, and therefore in proteins, having a knownsequence are well known. One of skill in the art using conventionaltechniques, such as PCR, may readily use the antigens and peptidesprovided herein to identify and isolate other similar proteins. Suchmethods are routine and not considered to require undue experimentation,given the information provided herein. For example, variations can bemade using oligonucleotide-mediated site-directed mutagenesis (Carter,P. et al., 1986; Zoller, M. J. et al., 1987), cassette mutagenesis(Wells, J. A. et al., 1985), restriction selection mutagenesis (Wells etal., 1986), PCR mutagenesis, or other known techniques can be performedon the cloned DNA to produce the peptide of the invention.

The polypeptide according to the present invention may be used for thedetection of the organism or organisms in a sample containing theseorganisms or proteins or antigens, including fragments thereof.Preferably such detection is for diagnosis, more preferable for thediagnosis of a disease, most preferably for the diagnosis of a diseaserelated or linked to the presence or abundance of Gram-negativebacteria, especially bacteria selected from the group comprisingpathogenic Klebsiella species, especially K. pneumoniae including thethree subspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca,K. planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca.

The nucleic acids according to the present invention can also be usedfor the diagnosis or detection of organisms or organisms in a sample,whereby the organisms are preferably the same ones as disclosed inconnection with the use of the polypeptides according to the presentinvention and the antibody according to the present invention,respectively. Basically, it is within the skills of the person of theart to design and practice such diagnosis and detection assays andmethods, respectively, in the light of the present disclosure. Morepreferably such diagnosis or detection uses primers or probes tospecifically interact with the nucleic acid molecules according to thepresent invention. The length and design of such primers and probes,respectively, varies depending on the particular method or diagnosispracticed. Using, in a preferred embodiment, a primer for, e.g., a PCRbased detection or diagnosis system, i.e. method or assay, the length ofthe primer will range from about 10 nucleotides to about 30 nucleotidesand more preferably from about 16 to 25 nucleotides. In case of a probebased detection or diagnosis system the length of the probe ispreferably about the same as specified for the primer based system.Additionally, in case of a probe based system, the probe will comprise amoiety which allows its detection, either directly or indirectly. Suchmoiety for direct detection can be a radioactive label or a fluorescencelabel as known to the ones skilled in the art. Such moiety for indirectdetection can be a biotin or any other moiety which mediates interactionwith a further compound which in turn is labelled so as to allow itsdetection.

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of thepolypeptides according to the present invention and more preferablyantigens and fragments thereof of the present invention in cells andtissues, including determination of normal and abnormal levels. Thus,for instance, a diagnostic assay in accordance with the invention fordetecting over-expression of the polypeptides according to the presentinvention compared to normal control tissue samples may be used todetect the presence of an infection, for example, and to identify theinfecting organism. Assay techniques that can be used to determinelevels of such polypeptides in a sample derived from a host are wellknown to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays. Among these, ELISA and Western Blot analysis frequentlyare preferred. An ELISA assay initially comprises preparing an antibodyspecific to one of the polypeptides according to the present invention,preferably a monoclonal antibody. In addition, a reporter antibodygenerally is prepared which binds to the monoclonal antibody. Thereporter antibody is attached to a detectable reagent such asradioactive, fluorescent or enzymatic reagent, such as horseradishperoxidase enzyme. One or several of the polypeptides according to thepresent invention and more preferably an antigen and fragment thereofaccording to the present invention may be immobilised on ELISA platesfor detection of reactive antibodies in sera of patients or subjects tobe tested.

A Western blot assay initially separates the polypeptides according tothe present invention individually or in combination bySDS-polyacrylamide gelelectrophoresis and which subsequently aretransferred and immobilised onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. Together with a reporterantibody reactive antibodies can be detected. The reporter antibody isattached to a detectable reagent such as radioactive, fluorescent orenzymatic reagent, such as horseradish peroxidase enzyme.

The polypeptides according to the present invention or the nucleic acidmolecules according to the present invention or primers or probesdirected thereto as described herein, may also be used for the purposeof or in connection with an array. In case of the nucleic acid moleculeaccording to the present invention and the primers and probes directedthereagainst, the length of the probes and the primer, can alsopreferably be in the range from about 25 to about 75 nucleotides, morepreferably from about 35 to about 50 nucleotides. More particularly, atleast one of the polypeptides according to the present invention may beimmobilized on a support. Said support typically comprises a variety ofthe polypeptides according to the present invention and/or antigens andfragments thereof whereby the variety may be created by using one orseveral of the antigens and fragments thereof according to the presentinvention and/or antigens and fragments thereof being different. Thecharacterizing feature of such array as well as of any array in generalis the fact that at a distinct or predefined region or position on saidsupport or a surface thereof, a distinct polypeptide is immobilized.Because of this any activity at a distinct position or region of anarray can be correlated with a specific polypeptide. The number ofdifferent polypeptides and more preferably different antigens andfragments thereof immobilized on a support may range from as little as10 to several 1,000 different polypeptides and antigens and fragmentsthereof, respectively. The density of said molecules per cm² is in apreferred embodiment as little as 10 per cm² to at least 400 differentof such polypeptides per cm² and more particularly at least 1,000different of such polypeptides and more preferably different antigensand fragments thereof per cm². What is said herein about theimmobilization of the polypeptides according to the present inventionand their use, is also applicable to the nucleic acid molecules and theprimers and probes, respectively, directed thereagainst, as will beacknowledged by the ones skilled in the art.

The manufacture of such arrays is known to the one skilled in the artand, for example, described in U.S. Pat. No. 5,744,309. The arraypreferably comprises a planar, porous or non-porous solid support havingat least a first surface. The polypeptides according to the presentinvention are immobilized on said surface. Preferred support materialsare, among others, glass or cellulose. It is also within the presentinvention that the array is used for any of the diagnostic applicationsdescribed herein. Apart from the polypeptides according to the presentinvention also the nucleic acid molecules according to the presentinvention may be used for the generation of an array as described abovewhich, in principle, can be used for any of the purposes disclosed forthe array containing polypeptides. This applies as well to an array madeof antibodies, preferably monoclonal antibodies as, among others,described herein.

In a further aspect the present invention relates to an antibodydirected to any of polypeptides according to the present invention,derivatives, fragments, variants, active fragments and active variantsthereof according to the present invention. The present inventionincludes, for example, monoclonal and polyclonal antibodies, chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of a Fab expression library. It is within the present inventionthat the antibody may be chimeric, i.e. that different parts thereofstem from different species or at least the respective sequences aretaken from different species.

Such antibodies in general and in particular directed against theantigens and fragments thereof corresponding to a sequence of thepresent invention can be obtained by direct injection of a polypeptideaccording to the present invention into an animal or by administeringsaid polypeptide to an animal, preferably a non-human. The antibody soobtained will then bind said polypeptide itself. In this manner, even asequence encoding only a fragment said polypeptide can be used togenerate antibodies binding the whole native polypeptides according tothe present invention. Such antibodies can then be used to isolate thepolypeptide according to the present invention from tissue expressingantigens and fragments thereof. It will be understood by the onesskilled in the art that this procedure is also applicable to thefragments, variants, active fragments and active variants thereof ofsaid polypeptides.

For preparation of monoclonal antibodies, any technique known in theart, which provides antibodies produced by continuous cell line culturescan be used (as described originally in (Kohler, G. et al., 1975)).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic antigens and fragments thereof in their diverse embodimentsaccording to this invention. Also, transgenic mice, or other organismssuch as other mammals, may be used to express humanized antibodies tothe polypeptides according to the present invention.

Still another aspect of the invention relates to a hybridoma cell linewhich produces the antibody of the invention.

Hybridoma cell lines expressing desirable monoclonal antibodies aregenerated by well-known conventional techniques. The hybridoma cell canbe generated by fusing a normal-activated, antibody-producing B cellwith a myeloma cell. In the context of the present invention thehybridoma cell is able to produce an antibody specifically binding tothe antigen of the invention.

Similarly, desirable high titer antibodies are generated by applyingknown recombinant techniques to the monoclonal or polyclonal antibodiesdeveloped to these antigens (see, e.g., PCT Patent Application No.PCT/GB85/00392; British Patent Application Publication No. GB2188638A;Amit, A. G. et al., 1986; Queen, C. et al., 1989; PCT Patent ApplicationNo. WO90/07861; Riechmann, L. et al., 1988; Huse, W. D. et al., 1988).

Alternatively, phage display technology or ribosomal display could beutilized to select antibody genes with binding activities towards thepolypeptides according to the present invention either from repertoiresof PCR amplified v-genes of lymphocytes from humans screened forpossessing respective target antigens or from naïve libraries(McCafferty, J. et al., 1990); (Marks, J. et al., 1992). The affinity ofthese antibodies can also be improved by chain shuffling (Clackson, T.et al., 1991).

If two antigen binding domains are present, each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides according to the present invention byattachment of the antibody to a solid support for isolation and/orpurification by affinity chromatography.

Thus, among others, antibodies against the polypeptides according to thepresent invention may be employed to inhibit and/or treat infections,particularly bacterial infections and especially infections arising frompathogenic Klebsiella species, especially K. pneumoniae including thethree subspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca,K. planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca.

The polypeptides according to the present invention and morespecifically antigens and fragments thereof in their diverse embodimentsinclude antigenically, epitopically or immunologically equivalentderivatives, which form a particular aspect of this invention. The term“antigenically equivalent derivative” as used herein encompasses suchpolypeptide according to the present invention or its equivalent whichwill be specifically recognized by certain antibodies which, when raisedto said polypeptide, interfere with the interaction between pathogen andmammalian host. The term “immunologically equivalent derivative” as usedherein encompasses a peptide or its equivalent which when used in asuitable formulation to raise antibodies in a vertebrate, the antibodiesact to interfere with the interaction between pathogen and mammalianhost.

The polypeptides according to the present invention and morespecifically the antigens and fragments thereof in their diverseembodiments, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof can be used as an antigen toimmunize a mouse or other animal such as a rat or chicken. The fusionprotein may provide stability to the polypeptide according to thepresent invention. Such polypeptide may be associated, for example byconjugation, with an immunogenic carrier protein, for example bovineserum albumin (BSA) or keyhole limpet haemocyanin (KLH). Alternatively,an antigenic peptide comprising multiple copies of the polypeptideaccording to the present invention and more preferably an antigen andfragments thereof, or an antigenically or immunologically equivalentantigen and fragments thereof, may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably the antibody or derivative thereof is modified to make itless immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized”, wherein thecomplementarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in (Jones, P. et al., 1986) or (Tempest, P. et al., 1991).

The use of a nucleic acid molecule according to the present invention ingenetic immunization will preferably employ a suitable delivery methodsuch as direct injection of plasmid DNA into muscle, delivery of DNAcomplexed with specific protein carriers, coprecipitation of DNA withcalcium phosphate, encapsulation of DNA in various forms of liposomes,particle bombardment (Tang, D. et al., 1992), (Eisenbraun, M. et al.,1993) and in vivo infection using cloned retroviral vectors (Seeger, C.et al., 1984).

In a further aspect the present invention relates to a peptide bindingto any of the polypeptides according to the present invention, and amethod for the preparation of such peptides whereby the method ischaracterized by the use of said polypeptide and the basic steps areknown to the one skilled in the art.

Such peptides may be generated by using methods according to the stateof the art such as phage display or ribosome display. In case of phagedisplay, basically a library of peptides is generated, in form ofphages, and this kind of library is contacted with the target molecule,in the present case a polypeptide according to the present invention.Those peptides binding to the target molecule are subsequently removed,preferably as a complex with the target molecule, from the respectivereaction. It is known to the one skilled in the art that the bindingcharacteristics, at least to a certain extent, depend on theparticularly realized experimental set-up such as the salt concentrationand the like. After separating those peptides binding to the targetmolecule with a higher affinity or a bigger force, from the non-bindingmembers of the library, and optionally also after removal of the targetmolecule from the complex of target molecule and peptide, the respectivepeptide(s) may subsequently be characterised. Prior to thecharacterisation optionally an amplification step is realized such as,e.g. by propagating the peptide encoding phages. The characterisationpreferably comprises the sequencing of the target binding peptides.Basically, the peptides are not limited in their lengths, howeverpreferably peptides having a length from about 8 to 20 amino acids arepreferably obtained in the respective methods. The size of the librariesmay be about 10² to 10¹⁸, preferably 10⁸ to 10¹⁵ different peptides,however, is not limited thereto. In a preferred embodiment such peptidesare high-affinity binding peptides. In an even more preferred embodimentthe peptides are peptide aptamers.

A particular form of target binding peptides as described above, are theso-called “anticalines” which are, among others, described in Germanpatent application DE 19742706. In so far, the present invention is alsorelated to peptides specifically binding to the polypeptides accordingto the present invention and the use thereof for any of the therapeuticand diagnostic applications described herein, preferably for antibodies.

In a further aspect the present invention relates to functional nucleicacids interacting with any of the polypeptides according to the presentinvention, and a method for the preparation of such functional nucleicacids whereby the method is characterized by the use of the polypeptidesaccording to the present invention and the basic steps are known to theone skilled in the art. The functional nucleic acids are preferablyaptamers and spiegelmers. In so far, the present invention is alsorelated to aptamers and spiegelmers specifically binding to thepolypeptides according to the present invention and the use thereof forany of the therapeutic and diagnostic applications described herein,preferably for antibodies.

Aptamers are D-nucleic acids, which are either single stranded or doublestranded and which specifically interact with a target molecule. Thepreparation or selection of aptamers is, e.g. described in Europeanpatent EP 0 533 838. Basically the following steps are realized. First,a mixture of nucleic acids, i.e. potential aptamers, is provided wherebyeach nucleic acid typically comprises a segment of several, preferablyat least eight subsequent randomised nucleotides. This mixture issubsequently contacted with the target molecule whereby the nucleicacid(s) bind to the target molecule, such as based on an increasedaffinity towards the target or with a bigger force thereto, compared tothe candidate mixture. The binding nucleic acid(s) are/is subsequentlyseparated from the remainder of the mixture. Optionally, the thusobtained nucleic acid(s) is amplified using, e.g. polymerase chainreaction. These steps may be repeated several times giving at the end amixture having an increased ratio of nucleic acids specifically bindingto the target from which the final binding nucleic acid is thenoptionally selected. These specifically binding nucleic acid(s) arereferred to as aptamers. It is obvious that at any stage of the methodfor the generation or identification of the aptamers samples of themixture of individual nucleic acids may be taken to determine thesequence thereof using standard techniques. It is within the presentinvention that the aptamers may be stabilized such as, e.g., byintroducing defined chemical groups which are known to the one skilledin the art of generating aptamers. Such modification may for examplereside in the introduction of an amino group at the 2′-position of thesugar moiety of the nucleotides. Aptamers are currently used astherapeutic agents. However, it is also within the present inventionthat the thus selected or generated aptamers may be used for targetvalidation and/or as lead substance for the development ofpharmaceutical compositions, preferably of pharmaceutical compositionsbased on small molecules. This is actually done by a competition assaywhereby the specific interaction between the target molecule and theaptamer is inhibited by a candidate drug whereby upon replacement of theaptamer from the complex of target and aptamer it may be assumed thatthe respective drug candidate allows a specific inhibition of theinteraction between target and aptamer, and if the interaction isspecific, said candidate drug will, at least in principle, be suitableto block the target and thus decrease its biological availability oractivity in a respective system comprising such target. The thusobtained small molecule may then be subject to further derivatisationand modification to optimise its physical, chemical, biological and/ormedical characteristics such as toxicity, specificity, biodegradabilityand bioavailability.

Spiegelmers and their generation or preparation is based on a similarprinciple. The preparation of spiegelmers is described in internationalpatent application WO 98/08856. Spiegelmers are L-nucleic acids, whichmeans that they are composed of L-nucleotides rather than D-nucleotidesas aptamers are. Spiegelmers are characterized by the fact that theyhave a very high stability in biological systems and, comparable toaptamers, specifically interact with the target molecule against whichthey are directed. In the process of generating spiegelmers, aheterogeneous population of D-nucleic acids is created and thispopulation is contacted with the optical antipode of the targetmolecule, in the present case for example with the D-enantiomer of thenaturally occurring L-enantiomer of the antigens and fragments thereofaccording to the present invention. Subsequently, those D-nucleic acidsare separated which do not interact with the optical antipode of thetarget molecule. But those D-nucleic acids interacting with the opticalantipode of the target molecule are separated, optionally identifiedand/or sequenced and subsequently the corresponding L-nucleic acids aresynthesized based on the nucleic acid sequence information obtained fromthe D-nucleic acids. These L-nucleic acids, which are identical in termsof sequence with the aforementioned D-nucleic acids interacting with theoptical antipode of the target molecule, will specifically interact withthe naturally occurring target molecule rather than with the opticalantipode thereof. Similar to the method for the generation of aptamersit is also possible to repeat the various steps several times and thusto enrich those nucleic acids specifically interacting with the opticalantipode of the target molecule.

In a further aspect the present invention relates to functional nucleicacids interacting with any of the nucleic acid molecules according tothe present invention, and a method for the preparation of suchfunctional nucleic acids whereby the method is characterized by the useof the nucleic acid molecules and their respective sequences accordingto the present invention and the basic steps are known to the oneskilled in the art. The functional nucleic acids are preferablyribozymes, antisense oligonucleotides and siRNA. In so far, the presentinvention is also related to this kind of functional nucleic acidspecifically binding to the polypeptides according to the presentinvention and the use thereof for any of the therapeutic and diagnosticapplications described herein, preferably for antibodies.

Ribozymes are catalytically active nucleic acids, which preferablyconsist of RNA, which basically comprises two moieties. The first moietyshows a catalytic activity whereas the second moiety is responsible forthe specific interaction with the target nucleic acid, in the presentcase the nucleic acid coding for the polypeptides according to thepresent invention. Upon interaction between the target nucleic acid andthe second moiety of the ribozyme, typically by hybridisation andWatson-Crick base pairing of essentially complementary stretches ofbases on the two hybridising strands, the catalytically active moietymay become active which means that it catalyses, either intramolecularlyor intermolecularly, the target nucleic acid in case the catalyticactivity of the ribozyme is a phosphodiesterase activity. Subsequently,there may be a further degradation of the target nucleic acid, which inthe end results in the degradation of the target nucleic acid as well asthe protein derived from the said target nucleic acid. Ribozymes, theiruse and design principles are known to the one skilled in the art, and,for example described in (Doherty, E. et al., 2001) and (Lewin, A. etal., 2001).

The activity and design of antisense oligonucleotides for thepreparation of a pharmaceutical composition and as a diagnostic agent,respectively, is based on a similar mode of action. Basically, antisenseoligonucleotides hybridise based on base complementarity, with a targetRNA, preferably with a mRNA, thereby activating RNase H. RNase H isactivated by both phosphodiester and phosphorothioate-coupled DNA.Phosphodiester-coupled DNA, however, is rapidly degraded by cellularnucleases with the exception of phosphorothioate-coupled DNA. Theseresistant, non-naturally occurring DNA derivatives do not inhibit RNaseH upon hybridisation with RNA. In other words, antisense polynucleotidesare only effective as DNA RNA hybrid complexes. Examples for this kindof antisense oligonucleotides are described, among others, in U.S. Pat.No. 5,849,902 and U.S. Pat. No. 5,989,912. In other words, based on thenucleic acid sequence of the target molecule which in the present caseare the nucleic acid molecules for the antigens and fragments thereofaccording to the present invention, either from the target protein fromwhich a respective nucleic acid sequence may in principle be deduced, orby knowing the nucleic acid sequence as such, particularly the mRNA,suitable antisense oligonucleotides may be designed base on theprinciple of base complementarity.

Particularly preferred are antisense-oligonucleotides, which have ashort stretch of phosphorothioate DNA (3 to 9 bases). A minimum of 3 DNAbases is required for activation of bacterial RNase H and a minimum of 5bases is required for mammalian RNase H activation. In these chimericoligonucleotides there is a central region that forms a substrate forRNase H that is flanked by hybridising “arms” comprised of modifiednucleotides that do not form substrates for RNase H. The hybridisingarms of the chimeric oligonucleotides may be modified such as by2″-O-methyl or 2′-fluoro. Alternative approaches used methylphosphonateor phosphoramidate linkages in said arms. Further embodiments of theantisense oligonucleotide useful in the practice of the presentinvention are P-methoxyoligonucleotides, partialP-methoxyoligodeoxy-ribonucleotides or P-methoxyoligodeoxy-ribonucleotides.

Of particular relevance and usefulness for the present invention arethose antisense oligonucleotides as more particularly described in theabove two mentioned US patents. These oligonucleotides contain nonaturally occurring 5″=>3′-linked nucleotides. Rather theoligonucleotides have two types of nucleotides:2′-deoxyphosphorothioate, which activate RNase H, and 2′-modifiednucleotides, which do not. The linkages between the 2′-modifiednucleotides can be phosphodiesters, phosphorothioate orP-ethoxyphosphodiester. Activation of RNase H is accomplished by acontiguous RNase H-activating region, which contains between 3 and 52′-deoxyphosphorothioate nucleotides to activate bacterial RNase H andbetween 5 and 10 2′-deoxyphosphorothioate nucleotides to activateeukaryotic and, particularly, mammalian RNase H.

Protection from degradation is accomplished by making the 5′ and 3′terminal bases highly nuclease resistant and, optionally, by placing a3′ terminal blocking group.

More particularly, the antisense oligonucleotide comprises a 5′ terminusand a 3′ terminus; and from position 11 to 59 5′=>3′-linked nucleotidesindependently selected from the group consisting of 2′-modifiedphosphodiester nucleotides and 2′-modified P-alkyloxyphosphotriesternucleotides; and wherein the 5′-terminal nucleoside is attached to anRNase H-activating region of between three and ten contiguousphosphorothioate-linked deoxyribonucleotides, and wherein the3″-terminus of said oligonucleotide is selected from the groupconsisting of an inverted deoxyribonucleotide, a contiguous stretch ofone to three phosphorothioate 2′-modified ribonucleotides, a biotingroup and a P-alkyloxyphosphotriester nucleotide.

Also an antisense oligonucleotide may be used wherein not the 5′terminal nucleoside is attached to an RNase H-activating region but the3′ terminal nucleoside as specified above. Also, the 5′ terminus isselected from the particular group rather than the 3′ terminus of saidoligonucleotide.

The nucleic acids as well as the polypeptides according to the presentinvention in their diverse embodiments may be used as or for thepreparation of pharmaceutical compositions, especially vaccines.Preferably such pharmaceutical composition, preferably vaccine is, forthe prevention or treatment of diseases caused by, related to orassociated with Klebsiella species, preferably pathogenic Klebsiella,especially K. pneumoniae including the three subspecies pneumoniae,ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena,and K. ornithinolytica, and more preferably from K. pneumoniae or K.oxytoca. In so far another aspect of the invention relates to a methodfor inducing an immunological response in an individual, particularly amammal, which comprises inoculating the individual with the polypeptidesaccording to the present invention in their diverse embodiments, or afragment or variant thereof, adequate to produce antibodies to protectsaid individual from infection by the above microorganisms.

Yet another aspect of the invention relates to a method of inducing animmunological response in an individual which comprises, through genetherapy or otherwise, delivering a nucleic acid molecule according tothe present invention, preferably functionally encoding antigens andfragments thereof in their diverse embodiments, for expressing thepolypeptide according to the present invention in vivo in order toinduce an immunological response to produce antibodies or a cellmediated T cell response, either cytokine-producing T cells or cytotoxicT cells, to protect said individual from disease, whether that diseaseis already established within the individual or not. One-way ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable of having inducedwithin it an immunological response, induces an immunological responsein such host, wherein the composition comprises recombinant DNA whichcodes for and expresses at least one of the polypeptides according tothe present invention in their diverse embodiments. The immunologicalresponse may be used therapeutically or prophylactically and may takethe form of antibody immunity or cellular immunity such as that arisingfrom CTL or CD4+ T cells.

The polypeptides according to the present invention in their diverseembodiments may be fused with a co-protein which may not by itselfproduce antibodies, but is capable of stabilizing the first protein andproducing a fused protein which will have immunogenic and protectiveproperties. This fused recombinant protein preferably further comprisesan antigenic co-protein, such as Glutathione-S-transferase (GST) orbeta-galactosidase, relatively large co-proteins which solubilise theprotein and facilitate production and purification thereof. Moreover,the co-protein may act as an adjuvant in the sense of providing ageneralized stimulation of the immune system. The co-protein may beattached to either the amino or carboxy terminus of the first protein.

Also provided by this invention are methods using the nucleic acidmolecule according to the present invention in their diverse embodimentsin such genetic immunization experiments in animal models of infectionwith any of the Klebsiella species described herein, especially K.pneumoniae including the three subspecies pneumoniae, ozaenae andrhinoscleromatis, K. oxytoca, K. planticola, K. terrigena, and K.ornithinolytica, and more preferably from K. pneumoniae or K. oxytoca.Such molecules will be particularly useful for identifying proteinepitopes able to provoke a prophylactic or therapeutic immune response.This approach can allow for the subsequent preparation of monoclonalantibodies of particular value from the requisite organ of the animalsuccessfully resisting or clearing infection for the development ofprophylactic agents or therapeutic treatments of the Klebsiella speciesdescribed herein and especially K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca, infection in mammals, particularlyhumans.

The polypeptides according to the present invention in their diverseembodiments may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage and thus damaged tissue include wounds in skinor connective tissue and mucosal tissues caused e.g. by viral infection(esp. respiratory, such as the flu) mechanical, chemical or thermaldamage or by implantation of indwelling devices, or wounds in the mucousmembranes, such as the mouth, mammary glands, urethra or vagina.

The present invention also includes a vaccine formulation, whichcomprises one or several of polypeptides according to the presentinvention in their diverse embodiments together with one or moresuitable carriers and/or excipients. The pharmaceutically acceptablecarriers and/or excipients useful in this invention are conventional andmay include buffers, stabilizers, diluents, preservatives, andsolubilizers. Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 15th Edition (1975), describes compositionsand formulations suitable for pharmaceutical delivery of the(poly)peptides herein disclosed. In general, the nature of the carrieror excipients will depend on the particular mode of administration beingemployed. For instance, parenteral formulations usually compriseinjectable fluids that include pharmaceutically and physiologicallyacceptable fluids such as water, physiological saline, balanced saltsolutions, aqueous dextrose, glycerol or the like as a vehicle. Forsolid compositions (e.g. powder, pill, tablet, or capsule forms),conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Since said polypeptides according to the present invention may be brokendown in the stomach, they are preferably administered parenterally,including, for example, administration that is subcutaneous,intramuscular, intravenous, intradermal, intranasal or transdermal.Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the body fluid, preferably the blood, of the individual; andaqueous and non-aqueous sterile suspensions which may include suspendingagents or thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampoules andvials, and may be stored in a freeze-dried condition requiring only theaddition of the sterile liquid carrier immediately prior to use. Thevaccine formulation may also include adjuvant systems for enhancing theimmunogenicity of the formulation, such as oil-in-water systems andother systems known in the art. The dosage will depend on the specificactivity of the vaccine and can be readily determined by routineexperimentation.

According to another aspect, the present invention relates to apharmaceutical composition comprising one or several of the polypeptidesaccording to the present invention in their diverse embodiments for thevarious Klebsiella species described herein and especially K. pneumoniaeincluding the three subspecies pneumoniae, ozaenae and rhinoscleromatis,K. oxytoca, K. planticola, K. terrigena, and K. ornithinolytica, andmore preferably from K. pneumoniae or K. oxytoca. Such a pharmaceuticalcomposition may comprise one, preferably at least two or more of saidpolypeptides against said Klebsiella species. Optionally, suchpolypeptides may also be combined with antigens against even furtherpathogens in a combination pharmaceutical composition. Preferably, saidpharmaceutical composition is a vaccine for preventing or treating aninfection caused by a Klebsiella species, more preferably a pathogenicKlebsiella species such as K. pneumoniae including the three subspeciespneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K.terrigena, and K. ornithinolytica, and more preferably from K.pneumoniae or K. oxytoca and/or other pathogens against which theantigens have been included in the vaccine.

According to a further aspect, the present invention relates to apharmaceutical composition comprising a nucleic acid molecule accordingto the present invention. Such a pharmaceutical composition may compriseone or more nucleic acid molecules according to the present inventionencoding a polypeptide according to the present invention. Optionally,such nucleic acid molecules encoding the polypeptides according to thepresent invention are combined with nucleic acid molecules encodingantigens against other pathogens in a combination pharmaceuticalcomposition. Preferably, said pharmaceutical composition is a vaccinefor preventing or treating an infection caused by Klebsiella species,more preferably pathogenic Klebsiella species as disclosed herein,especially K. pneumoniae including the three subspecies pneumoniae,ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena,and K. ornithinolytica, and more preferably from K. pneumoniae or K.oxytoca, and/or other pathogens against which the antigens have beenincluded in the vaccine.

The pharmaceutical composition may contain any suitable auxiliarysubstances, such as buffer substances, stabilisers or further activeingredients, especially ingredients known in connection ofpharmaceutical composition and/or vaccine production.

In a preferred embodiment the pharmaceutical composition furthercomprises an immunostimulatory substance such as an adjuvant. Theadjuvant can be selected based on the method of administration and mayinclude polycationic substances, especially polycationic peptides,immunostimulatory nucleic acids molecules, preferably immunostimulatoryoligo-deoxynucleotides (ODNs), especially Oligo(dIdC)₁₃, peptidescontaining at least two LysLeuLys motifs, especially peptideKLKLLLLLKLK, alum, mineral oil-based adjuvants such as Freund's completeadjuvant and Freund's incomplete adjuvant, neuroactive compounds,especially human growth hormone, or any combination of one or more ofthe above mentioned adjuvants. Other suitable adjuvants may be selectedfrom the group consisting of Montanide incomplete Seppic adjuvant suchas ISA, oil in water emulsion adjuvants such as the Ribi adjuvantsystem, syntax adjuvant formulation containing muramyl dipeptide, oraluminum salt adjuvants or combinations thereof. Preferably, theadjuvant is IC31® (Intercell; a synthetic adjuvant comprising thepeptide motif KLK as described in WO 02/32451 and an oligonucleotide asdescribed in WO 01/93905).

The term “Oligo(dIdC)₁₃” as used in the present invention means aphosphodiester substituted single-stranded ODN containing 13 deoxy(inosine-cytosine) motifs, also defined by the term [oligo-d(IC)₁₃]. Theexact sequence is5′-dIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdC-3′.Oligo(dIdC)₁₃ can also be defined by the terms (oligo-dIC₂₆);oligo-dIC_(26-mer); oligo-deoxy IC, 26-mer; or oligo-dIC, 26-mer, asspecified for example in WO 01/93903 and WO 01/93905.

It is also within the scope of the present invention that thepharmaceutical composition, especially vaccine, comprises apart from oneor several of he polypeptides according to the present invention intheir diverse embodiments and/or nucleic acid molecules coding thereofwhich are also in accordance with the present invention, other compoundswhich are biologically or pharmaceutically active. Preferably, thevaccine composition comprises at least one polycationic peptide. Thepolycationic compound(s) to be used according to the present inventionmay be any polycationic compound, which shows the characteristic effectsaccording to the WO 97/30721. Preferred polycationic compounds areselected from basic polypeptides, organic polycations, basic polyaminoacids or mixtures thereof. These polyamino acids should have a chainlength of at least 4 amino acid residues (WO 97/30721). Especiallypreferred are substances like polylysine, polyarginine and polypeptidescontaining more than 20%, especially more than 50% of basic amino acidsin a range of more than 8, especially more than 20, amino acid residuesor mixtures thereof. Other preferred polycations and theirpharmaceutical compositions are described in WO 97/30721 (e.g.polyethyleneimine) and WO 99/38528. Preferably these polypeptidescontain between 20 and 500 amino acid residues, especially between 30and 200 residues.

These polycationic compounds may be produced chemically or recombinantlyor may be derived from natural sources.

Cationic (poly)peptides may also be anti-microbial with properties asreviewed in (Ganz, T., 1999). These (poly)peptides may be of prokaryoticor animal or plant origin or may be produced chemically or recombinantly(WO 02/13857). Peptides may also belong to the class of defensins (WO02/13857). Sequences of such peptides can be, for example, found in theAntimicrobial Sequences Database under the following internet address:http://www.bbcm.univ.trieste.it/˜tossi/pag2.html

Such host defence peptides or defensives are also a preferred form ofthe polycationic polymer according to the present invention. Generally,a compound allowing as an end product activation (or down-regulation) ofthe adaptive immune system, preferably mediated by APCs (includingdendritic cells) is used as polycationic polymer.

Especially preferred for use as polycationic substances in the presentinvention are cathelicidin derived antimicrobial peptides or derivativesthereof (International patent application WO 02/13857, incorporatedherein by reference), especially antimicrobial peptides derived frommammalian cathelicidin, preferably from human, bovine or mouse.

Polycationic compounds derived from natural sources include HIV-REV orHIV-TAT (derived cationic peptides, antennapedia peptides, chitosan orother derivatives of chitin) or other peptides derived from thesepeptides or proteins by biochemical or recombinant production. Otherpreferred polycationic compounds are cathelin or related or derivedsubstances from cathelin. For example, mouse cathelin is a peptide,which has the amino acid sequenceNH₂—RLAGLLRKGGEKIGEKLKKIGOKIKNFFQKLVPQPE-COOH. Related or derivedcathelin substances contain the whole or parts of the cathelin sequencewith at least 15-20 amino acid residues. Derivations may include thesubstitution or modification of the natural amino acids by amino acids,which are not among the 20 standard amino acids. Moreover, furthercationic residues may be introduced into such cathelin molecules. Thesecathelin molecules are preferred to be combined with the antigen. Thesecathelin molecules surprisingly have turned out to be also effective asan adjuvant for an antigen without the addition of further adjuvants. Itis therefore possible to use such cathelin molecules as efficientadjuvants in vaccine formulations with or without furtherimmunactivating substances.

Another preferred polycationic substance to be used in accordance withthe present invention is a synthetic peptide containing at least 2KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids(International patent application WO 02/32451, incorporated herein byreference).

The pharmaceutical composition of the present invention may furthercomprise immunostimulatory nucleic acid(s). Immunostimulatory nucleicacids are e.g. neutral or artificial CpG containing nucleic acids, shortstretches of nucleic acids derived from non-vertebrates or in form ofshort oligonucleotides (ODNs) containing non-methylated cytosine-guaninedi-nucleotides (CpG) in a certain base context (e.g. described in WO96/02555). Alternatively, also nucleic acids based on inosine andcytidine as e.g. described in the WO 01/93903, or deoxynucleic acidscontaining deoxy-inosine and/or deoxyuridine residues (described in WO01/93905 and WO 02/095027, incorporated herein by reference) maypreferably be used as immunostimulatory nucleic acids in connection withthe present invention. Preferably, the mixtures of differentimmunostimulatory nucleic acids may be used according to the presentinvention.

It is also within the present invention that any of the aforementionedpolycationic compounds is combined with any of the immunostimulatorynucleic acids as aforementioned. Preferably, such combinations areaccording to the ones as described in WO 01/93905, WO 02/32451, WO01/54720, WO 01/93903, WO 02/13857, WO 02/095027 and WO 03/047602,incorporated herein by reference.

In addition or alternatively such vaccine composition may comprise apartfrom the polypeptides according to the present invention, and thenucleic acid molecules according to the present invention, preferablythe coding nucleic acid molecules according to the present invention, aneuroactive compound. Preferably, the neuroactive compound is humangrowth factor as, e.g. described in WO 01/24822. Also preferably, theneuroactive compound is combined with any of the polycationic compoundsand/or immunostimulatory nucleic acids as afore-mentioned.

Also, the pharmaceutical composition in accordance with the presentinvention is a pharmaceutical composition which comprises at least anyof the following compounds or combinations thereof: the nucleic acidmolecules according to the present invention, the polypeptides accordingto the present invention in their diverse embodiments, the vectoraccording to the present invention, the cells according to the presentinvention, the antibody according to the present invention, thefunctional nucleic acids according to the present invention and thebinding peptides such as the anticalines and high-affinity bindingpeptides and peptide aptamers, respectively, according to the presentinvention, any agonists and antagonists according to the presentinvention, preferably screened as described herein. In connectiontherewith any of these compounds may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of an antigen andfragments thereof of the invention and a pharmaceutically acceptablecarrier or excipient. Such carriers may include, but are not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol andcombinations thereof. The formulation should suit the mode ofadministration.

The composition may be used e.g. for immunization or treatment of asubject. The pharmaceutical composition encompasses at least one peptideof the invention; however, it may also contain a cocktail (i.e., asimple mixture) containing different peptides (including fragments andother variants) of the invention, optionally mixed with differentantigenic proteins or peptides of other pathogens. Such mixtures ofthese peptides, polypeptides, proteins or fragments or variants thereofare useful e.g. in the generation of desired antibodies to a widespectrum of Klebsiella isolates. The peptide(s) of the present inventionmay also be used in the form of a pharmaceutically acceptable salt.Suitable acids and bases which are capable of forming salts with thepeptides of the present invention are well known to those of skill inthe art, and include inorganic and organic acids and bases.

Still another aspect of the present invention is a pharmaceuticalcomposition containing a nucleic acid selected from the group consistingof:

(i) a nucleic acid of the invention and/or a nucleic acid complementarythereto, and(ii) optionally a pharmaceutically acceptable carrier or excipient.

The nucleic acid sequences, alone or in combination with other nucleicacid sequences encoding antigens or antibodies or directed to otherpathogenic microorganisms, may further be used as components of apharmaceutical composition. The composition may be used for immunizingor treating humans and/or animals with the disease caused by infectionwith Klebsiella, preferably K. pneumoniae including the three subspeciespneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K.terrigena, and K. ornithinolytica, and more preferably from K.pneumoniae or K. oxytoca. The pharmaceutically acceptable carrier orexcipient may be as defined above.

In another embodiment, the nucleic acid sequences of this invention,alone or in combination with nucleic acid sequences encoding otherantigens or antibodies from other pathogenic microorganisms, may furtherbe used in compositions directed to actively induce a protective immuneresponse to the pathogen in a subject. These components of the presentinvention are useful in methods for inducing a protective immuneresponse in humans and/or animals against infection with Klebsiella,preferably K. pneumoniae including the three subspecies pneumoniae,ozaenae and rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena,and K. ornithinolytica, and more preferably from K. pneumoniae or K.oxytoca.

For use in the preparation of the therapeutic or vaccine compositions,nucleic acid delivery compositions and methods are useful, which areknown to those of skill in the art. The nucleic acid of the inventionmay be employed in the methods of this invention or in the compositionsdescribed herein as DNA sequences, either administered as naked DNA, orassociated with a pharmaceutically acceptable carrier and provide for invivo expression of the antigen, peptide or polypeptide. So-called “nakedDNA” may be used to express the antigen, peptide or polypeptide of theinvention in vivo in a patient. (See, e.g., Cohen, J., 1993, whichdescribes similar uses of “naked DNA”). For example, “naked DNA”associated with regulatory sequences may be administered therapeuticallyor as part of the vaccine composition e.g., by injection.

Alternatively, a nucleic acid encoding the antigens or peptides of theinvention or a nucleic acid complementary thereto may be used within apharmaceutical composition, e.g. in order to express the antigens orpeptides or polypeptides of the invention in vivo, e.g., to induceantibodies.

A preferred embodiment of the invention relates to a pharmaceuticalcomposition, wherein the nucleic acid according to the invention iscomprised in a vector and/or a cell. Vectors and cells suitable in thecontext of the present invention are described above. Vectors areparticularly employed for a DNA vaccine. An appropriate vector fordelivery may be readily selected by one of skill in the art. Exemplaryvectors for in vivo gene delivery are readily available from a varietyof academic and commercial sources, and include, e.g., adeno-associatedvirus (International patent application No. PCT/US91/03440), adenovirusvectors (Kay, M. et al., 1994; Ishibashi, S. et al., 1993), or otherviral vectors, e.g., various poxviruses, vaccinia, etc. Recombinantviral vectors, such as retroviruses or adenoviruses, are preferred forintegrating the exogenous DNA into the chromosome of the cell.

The pharmaceutical compositions of the present invention may beadministered in any effective, convenient manner including, forinstance, administration by topical, oral, anal, vaginal, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal, intratrachealor intradermal routes among others.

In therapy or as a prophylactic, the active agent of the pharmaceuticalcomposition of the present invention may be administered to anindividual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition, preferably the pharmaceutical compositionmay be formulated for topical application, for example in the form ofointments, creams, lotions, eye ointments, eye drops, ear drops,mouthwash, impregnated dressings and sutures and aerosols, and maycontain appropriate conventional additives, including, for example,preservatives, solvents to assist drug penetration, and emollients inointments and creams. Such topical formulations may also containcompatible conventional carriers, for example cream or ointment bases,and ethanol or oleyl alcohol for lotions. Such carriers may constitutefrom about 1% to about 98% by weight of the formulation; more usuallythey will constitute up to about 80% by weight of the formulation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

In a preferred embodiment the pharmaceutical composition is a vaccinecomposition. Preferably, such vaccine composition is conveniently ininjectable form. Conventional adjuvants may be employed to enhance theimmune response. A suitable unit dose for vaccination with a proteinantigen is for adults between 0.02 to 3 μg antigen/per kg of body weightand for children between 0.2 to 10 μg antigen/per kg body weight, andsuch dose is preferably administered 1-3 times and with an interval of 2to 24 weeks.

An “effective amount” or “therapeutically effective amount” of anantigen, nucleic acid, vector, an antibody or a pharmaceuticalcomposition of the invention may be calculated as that amount capable ofexhibiting an in vivo effect, e.g. preventing or ameliorating a sign orsymptom of infection with Klebsiella, especially K. pneumoniae includingthe three subspecies pneumoniae, ozaenae and rhinoscleromatis, K.oxytoca, K. planticola, K. terrigena, and K. ornithinolytica, and morepreferably from K. pneumoniae or K. oxytoca. Such amounts may bedetermined by one of skill in the art.

With the indicated dose range, no adverse toxicological effects areexpected with the compounds of the invention, which would preclude theiradministration to suitable individuals.

In a further embodiment the present invention relates to diagnostic andpharmaceutical packs and kits comprising one or more containers filledwith one or more of the ingredients of the aforementioned compositionsof the invention. The ingredient(s) can be present in a useful amount,dosage, formulation or combination. Associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the preparation, use or sale of pharmaceuticals or biologicalproducts, reflecting approval by the agency of the preparation, use orsale of the product for human administration.

In connection with the present invention any disease related use asdisclosed herein such as, e.g., use of the pharmaceutical composition orvaccine, is particularly a disease or diseased condition which is causedby, linked or associated with Klebsiella, more preferably any pathogenicKlebsiella species and especially K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca. A disease related, caused orassociated with the bacterial infection to be prevented and/or treatedaccording to the present invention includes nosocomial infections.Common sites include the urinary tract, lower respiratory tract, biliarytract, and surgical wound sites. The spectrum of clinical syndromesincludes pneumonia, bacteraemia, thrombophlebitis, urinary tractinfection (UTI), cholecystitis, diarrhea, upper respiratory tractinfection, wound infection, osteomyelitis, and meningitis.

It is within the present invention that each and any of the symptoms,diseases, disorders or syndromes described herein which are eitherdirectly or indirectly linked to or arise from a contact of an organismsuch as any animal or human with a Klebsiella species, more preferably apathogenic Klebsiella species, and especially K. pneumoniae includingthe three subspecies pneumoniae, ozaenae and rhinoscleromatis, K.oxytoca, K. planticola, K. terrigena, and K. ornithinolytica, and morepreferably from K. pneumoniae or K. oxytoca are separately andindependently indications, diseases or disorders in the meaning of thepresent invention. Accordingly and just by means of illustration, adisease in the sense of the present application is septicaemia as wellas meningitis and osteomyelitis.

It is within the present invention that the disease for which thevarious compounds described herein can be used are also those diseaseswhere the polypeptide according to the present invention is expressed orany disease where the compounds described herein such as thepolypeptides according to the present invention, the vaccine, theantibody, and any aptamer and spiegelmer, respectively, are suitable forthe treatment and/or diagnosis thereof. Such potential use can arisefrom cross-reactivity and homology, respectively. It understood by theone skilled in the art that any disease described in connection with thepharmaceutical composition according to the present invention can besubject to the use of the pharmaceutical composition described herein,and vice versa.

Treatment in the context of the present invention refers to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) the targeted pathologiccondition or disorder. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein whom the disorder is to be prevented.

In a still further embodiment the present invention is related to ascreening method using any of the polypeptides according to the presentinvention or any of the nucleic acids according to the presentinvention. Screening methods as such are known to the one skilled in theart and can be designed such that an agonist or an antagonist isscreened. In connection with such screening method preferably anantagonist is screened which in the present case inhibits or preventsthe binding of any antigen and fragment thereof according to the presentinvention to an interaction partner. Such interaction partner can be anaturally occurring interaction partner or a non-naturally occurringinteraction partner.

The invention also provides a method for screening compounds to identifythose, which enhance (agonist) or block (antagonist) the function of thepolypeptides according to the present invention or of the nucleic acidmolecules of the present invention, such as its interaction with abinding molecule. The method of screening may involve high-throughput.

For example, to screen for agonists or antagonists, the interactionpartner of the nucleic acid molecule and nucleic acid, respectively,according to the present invention, maybe a synthetic reaction mix, acellular compartment, such as a membrane, cell envelope or cell wall, ora preparation of any thereof, may be prepared from a cell that expressesa molecule that binds to the polypeptides according to the presentinvention. The preparation is incubated with labelled forms of suchpolypeptides in the absence or the presence of a candidate molecule,which may be an agonist or antagonist. The ability of the candidatemolecule to bind the binding molecule is reflected in decreased bindingof the labelled ligand. Molecules which bind gratuitously, i.e., withoutinducing the functional effects of said polypeptides, are most likely tobe good antagonists. Molecules that bind well and elicit functionaleffects that are the same as or closely related to the polypeptidesaccording to the present invention are good agonists.

The functional effects of potential agonists and antagonists may bemeasured, for instance, by determining the activity of a reporter systemfollowing interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that ofpolypeptides according to the present invention or molecules that elicitthe same effects as said polypeptides. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabelled substrate converted into product, a reporter gene that isresponsive to changes in the functional activity of the polypeptidesaccording to the present invention, and binding assays known in the art.

Another example of an assay for antagonists is a competitive assay thatcombines the polypeptides according to the present invention and apotential antagonist with membrane-bound binding molecules, recombinantbinding molecules, natural substrates or ligands, or substrate or ligandmimetics, under appropriate conditions for a competitive inhibitionassay. The polypeptides according to the present invention can belabelled such as by radioactivity or a colorimetric compound, such thatthe molecule number of polypeptides according to the present inventionbound to a binding molecule or converted to product can be determinedaccurately to assess the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to polypeptides according to thepresent invention and thereby inhibit or extinguish its activity.Potential antagonists may also be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds tothe same sites on a binding molecule without inducing functionalactivity of the polypeptides according to the present invention.

Potential antagonists include a small molecule, which binds to andoccupies the binding site of the polypeptides according to the presentinvention thereby preventing binding to cellular binding molecules, suchthat normal biological activity is prevented. Examples of smallmolecules include but are not limited to small organic molecules,peptides or peptide-like molecules.

Other potential antagonists include antisense molecules (see (Okano, H.et al., 1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENEEXPRESSION; CRC Press, Boca Raton, Fla. (1988), for a description ofthese molecules).

Preferred potential antagonists include derivatives of the antigens andfragments thereof of the invention.

As used herein the activity of a polypeptide according to the presentinvention is its capability to bind to any of its interaction partner orthe extent of such capability to bind to its or any interaction partner.

In a particular aspect, the invention provides the use of thepolypeptides according to the present invention antigens and fragmentsthereof, nucleic acid molecules or inhibitors of the invention tointerfere with the initial physical interaction between a pathogen andmammalian host responsible for sequelae of infection. In particular themolecules of the invention may be used: i) in the prevention of adhesionof the Klebsiella species as disclosed herein and more preferably thepathogenic species thereof, especially K. pneumoniae including the threesubspecies pneumoniae, ozaenae and rhinoscleromatis, K. oxytoca, K.planticola, K. terrigena, and K. ornithinolytica, and more preferablyfrom K. pneumoniae or K. oxytoca to mammalian extracellular matrixproteins; ii) to block bacterial adhesion between mammalianextracellular matrix proteins and bacterial proteins which mediatetissue reaction; iii) or lead to evasion of immune defence; iv) to blockthe normal progression of pathogenesis in infections initiated otherthan by the implantation of in-dwelling devices or by other surgicaltechniques, e.g. through inhibiting nutrient acquisition.

Each of the DNA coding sequences provided herein may be used in thediscovery, development and/or preparation of antibacterial compounds.The encoded protein upon expression can be used as a target for thescreening of antibacterial drugs. Additionally, the DNA sequencesencoding the amino terminal regions of the encoded protein orShine-Delgarno or other translation facilitating sequences of therespective mRNA can be used to construct antisense sequences to controlthe expression of the coding sequence of interest.

The antagonists and agonists may be employed, for instance, to inhibitdiseases arising from infection with Klebsiella species, especially K.pneumoniae including the three subspecies pneumoniae, ozaenae andrhinoscleromatis, K. oxytoca, K. planticola, K. terrigena, and K.ornithinolytica, and more preferably from K. pneumoniae or K. oxytoca.

In a still further aspect the present invention is related to anaffinity device such affinity device comprises as least a supportmaterial and any of the polypeptides according to the present invention,which is attached to the support material. Because of the specificity ofsaid polypeptides for their target cells or target molecules or theirinteraction partners, said polypeptides allow a selective removal oftheir interaction partner(s) from any kind of sample applied to thesupport material provided that the conditions for binding are met. Thesample may be a biological or medical sample, including but not limitedto, fermentation broth, cell debris, cell preparation, tissuepreparation, organ preparation, blood, urine, lymph liquid, liquor andthe like.

The polypeptides according to the present invention may be attached tothe matrix in a covalent or non-covalent manner. Suitable supportmaterial is known to the one skilled in the art and can be selected fromthe group comprising cellulose, silicon, glass, aluminium, paramagneticbeads, starch and dextrane.

The present invention is further illustrated by the following Figures,Tables, Examples and the Sequence Listing, from which further features,embodiments and advantages may be taken. It is to be understood that thepresent examples are given by way of illustration only and not by way oflimitation of the disclosure.

In connection with the present invention

FIG. 1 shows the characterization of human sera as sources of pathogenspecific antibodies.

FIG. 2 shows the characterization of the libraries.

FIG. 3 shows the selection of bacterial cells by MACS using biotinylatedhuman IgGs.

FIG. 4 shows the PCR analysis to determine the gene distribution of aselected antigen in clinical isolates of the respective bacterialpathogen.

FIG. 5 shows examples for surface staining with epitope sera generatedin mice.

FIG. 6 shows the protection conferred by active immunization withselected K. pneumoniae antigens in a mouse lethality model, when CFA/IFAwas used as adjuvant.

FIG. 7 shows the protection conferred by active immunization withselected K. pneumoniae antigens in a mouse lethality model, when Alumwas used as adjuvant.

FIG. 8 shows the protection conferred by active immunization withselected K. pneumoniae antigens in a mouse lethality model, when IC31®was used as adjuvant.

FIG. 9 shows the protection conferred by passive immunization withpolyclonal rabbit sera raised against selected K. pneumoniae antigens ina mouse lethality model.

Table 1 shows the summary of all screens performed with genomic K.pneumoniae libraries and human serum.

Table 2 shows the strains used for gene distribution analysis.

Table 3 shows the summary of the gene distribution analysis for aselected number of antigens in various strains of the respectivebacterial species.

Table 4 shows the summary of the peptide ELISA with human sera.

Table 5 Surface staining with epitope sera generated in mice.

Table 6 shows independent Klebsiella sp. isolates used to amplify genesof interest.

Table 7 shows the sequences of the oligonucleotides used to amplifygenes of interest.

Table 8 Gene conservation of KPORF-13.

Table 9 Gene conservation of KPORF-21.

Table 10 Gene conservation of KPORF-32.

Table 11 Gene conservation of KPORF-37.

Table 12 Gene conservation of KPORF-38.

Table 13 Gene conservation of KPORF-39.

Table 14 Gene conservation of KPORF-60.

Table 15 Gene conservation of KPORF-65.

Table 16 Antigen fragments used for protection experiments.

The figures and tables to which it might be referred to in thespecification are described in the following in more details.

FIG. 1 shows the characterization of human sera by measuring antibodiesspecific for K. pneumoniae by immune assays. Total IgG antibody levelswere measured by standard ELISA. (A) total bacterial lysates preparedfrom K. pneumoniae strain Mich 61 and its respective capsule negativemutant. (B) total bacterial lysates prepared from K. pneumoniae strain708 and its respective capsule negative mutant. Serum samples fromhealthy individuals and patients with septicaemia were analyzed at twodifferent serum dilutions (1:1,000 and 1:5,000). P3536.2, P3495.2 andP3533.2 were convalescent sera obtained from patients after recoveringfrom sickness. Results of the sera selected for the four different poolsat a serum dilution of 1:1,000 are shown.

FIG. 2 (A) shows the fragment size distribution of the K. pneumoniaesmall fragment genomic library, KPL50 in pMAL4.31. After sequencingrandomly selected clones, sequences were trimmed (476) to eliminatevector residues and the numbers of clones with various genomic fragmentsizes were plotted. (B) shows the fragment size distribution of the K.pneumoniae large fragment genomic library, KPF300 in pMAL4.31. Sequencesof randomly selected clones were trimmed (425) and the numbers of cloneswith various genomic fragment sizes were plotted.

FIG. 3 (A) shows the MACS selection with the biotinylated human IgG poolwith sera from patients with septicaemia. The KPL50 library in pMAL9.1was screened with 10-20 μg biotinylated IgG. As negative control, noserum was added to the library cells for screening. Number of cellsselected after elution are shown. (B) shows the reactivity of specificclones (1-20) selected by bacterial surface display and Wt (pMAL9.1without insert) as analyzed by immunoblot analysis with the human serumIgG pool (PKp34-IgG) used for selection by MACS at a dilution of1:3,000. Asterisks indicate the clones detected as positive. As aloading control the same blot was also analyzed with antibodies directedagainst the platform protein LamB at a dilution of 1:5,000 (data notshown). (C) shows the MACS selection with the biotinylated human IgGpool with sera from patients with septicaemia (PKp35-IgG) and the KPF300library in pHIE11. (D) shows the reactivity of specific clones (1-20)selected by bacterial surface display and Wt (pHIE11 without insert) asanalyzed by immunoblot analysis with the human serum IgG pool(PKp35-IgG) used for selection by MACS at a dilution of 1:3,000.Asterisks indicate the clones detected as positive.

FIG. 4 shows an example for the PCR analysis for the gene distributionanalysis of one gene with the respective oligonucleotides and 46 K.pneumoniae strains. The predicted size of the PCR fragment derived fromantigen KPORF-54 from K. pneumoniae is 1040 bp. 1-46: strains orclinical isolates as shown in Table 2; −: no genomic DNA; +: genomic DNAof K. pneumoniae strain MGH78578.

FIG. 5 shows examples for surface staining with epitope sera generatedin mice. Efficient surface display of three antigens on K. pneumoniaeA5054 is shown. KPORF-28, KPORF-82 and KPORF-02 are examples forcategory “+”, 0-9%; “++”, 10-35% and “+++”, >36%. Percentage indicatesthe number of cells that showed a shift in the FACS analysis incomparison to cells incubated without immune sera.

FIGS. 6 (A), (B), (C), (D) and (E) show the protection achieved byactive immunization with selected K. pneumoniae antigens in a mouselethality model. CD-1 mice (10 mice per group) were immunizedsubcutaneously with recombinant antigens cloned from a K. pneumoniaestrain MHG78578 and challenged with K. pneumoniae (O1:K2) strain B5055.Survival was monitored for 14 days post-challenge. Mice were immunizedsubcutaneously with 50 μg recombinant protein adjuvanted with CFA/IFA orIFA. Mice immunized with PBS combined with CFA/IFA or IFA were used asnegative controls, while mice immunized with K. pneumoniae B5055 lysate(5 μg) served as a positive control. Mice were challengedintraperitoneally with 10³ CFU K. pneumoniae B5055. Numbers of survivingmice are plotted as percentage of total mice.

FIGS. 7 (A) and (B) show the protection conferred by active immunizationwith selected K. pneumoniae antigens in a mouse lethality model. CD-1mice (10 mice per group) were immunized subcutaneously with recombinantantigens cloned from a K. pneumoniae strain MHG78578 and challenged withK. pneumoniae (O1:K2) strain B5055. Survival was monitored for 14 dayspost-challenge. Mice were immunized subcutaneously with 50 μgrecombinant protein adjuvanted with Alum. Mice immunized with PBScombined with Alum were used as negative controls, while mice immunizedwith K. pneumoniae B5055 lysate (5 μg) combined with Alum served as apositive control. Mice were challenged intraperitoneally with 10³ CFU K.pneumoniae B5055. Numbers of surviving mice are plotted as a percentageof total mice.

FIGS. 8 (A) and (B) show the protection conferred by active immunizationwith selected K. pneumoniae antigens in a mouse lethality model. CD-1mice (10 mice per group) were immunized subcutaneously with recombinantantigens cloned from a K. pneumoniae strain MHG78578 and challenged withK. pneumoniae (O1:K2) strain B5055. Survival was monitored for 14 dayspost-challenge. Mice were immunized subcutaneously with 50 μgrecombinant protein adjuvanted with IC31®. Mice immunized with PBScombined with IC31® were used as negative controls, while mice immunizedwith K. pneumoniae B5055 lysate (5 μg) combined with IC31® served as apositive control. Mice were challenged intraperitoneally with 10³ CFU K.pneumoniae B5055. Numbers of surviving mice are plotted as a percentageof total mice.

FIGS. 9 (A) and (B) show the protection conferred by passiveimmunization with polyclonal rabbit sera raised against selected K.pneumoniae antigens in a mouse lethality model. CD-1 mice (10 mice pergroup) were immunized intraperitoneally with sera raised againstrecombinant antigens cloned from a K. pneumoniae strain MHG78578 andchallenged with K. pneumoniae (O1:K2) strain B5055. Survival wasmonitored for 14 days post-challenge. Mice immunized with PBS sera wereused as negative controls, while mice immunized with K. pneumoniae B5055lysate sera (5 μg) served as a positive control. Mice were challengedintraperitoneally with 10³ CFU K. pneumoniae B5055. Numbers of survivingmice are plotted as a percentage of total mice.

TABLE 1 Immunogenic proteins identified from K. pneumoniae by bacterialsurface display. Location of No. of identified SeqID Putative function(by Predicted selected Identified immunogenic (DNA, ORF homology)immunogenic aa* clones in Screen region (aa) Prot.) KPORF- NADH 11-27,35-47, 68-107, 2 H 180-226 17, 01 pyrophosphatase 113-122, 124-136, 204140-146, 152-164, 168-174, 183-201, 211-218, 228-243, 246-253 KPORF-Lactoylglutathione 13-31, 48-59, 69-91, 2 D  46-105 18, 02 lyase andrelated 109-115, 121-127 205 lyases KPORF- ABC-type Fe3+ 12-44, 49-95,102-145, 3 A 273-286 19, 03 transport system, 148-178, 184-229, 206permease component 233-244, 249-273, 292-299, 304-329, 334-348, 354-365,367-385, 394-426, 428-440, 444-487, 503-527, 531-539, 546-554, 556-584KPORF- Hypothetical 7-17, 22-32, 34-41, 2 G 211-269 20, 04transcriptional 55-77, 79-86, 93-111, 207 activator 118-126, 131-148,152-162, 165-177, 183-197, 213-220, 234-250, 253-262, 267-294 KPORF- 23SrRNA 22-29, 41-56, 58-66, 3 H  1-48 21, 05 pseudouridylate 79-88,94-121, 208 synthase 124-131, 134-157, 162-171, 173-180, 189-197,201-214, 216-224, 242-254, 257-270, 282-287, 290-302, 309-315, 320-325,341-355, 362-368, 372-378 KPORF- Glucosyltransferase 5-15, 18-35, 48-61,2 C 31-83 22, 06 MdoH 65-71, 112-119, 209 138-154, 157-169, 179-208,214-223, 226-232, 243-250, 256-262, 277-286, 289-296, 338-348, 352-363,370-376, 385-408, 420-436, 443-454, 462-483, 498-561, 563-592, 600-642,661-671, 673-709, 714-733, 748-754, 771-776, 798-806, 808-821, 823-839KPORF- trp-repressor binding 5-14, 21-26, 31-41, 3 H  97-158 23, 07protein 59-77, 101-115, 210 132-145, 147-156, 180-185, 188-197 KPORF-Putative 6-18, 23-43, 45-56, 2 G 521-583 24, 08 transcriptional 69-80,87-97, 112-123, 211 regulator 135-151, 164-171, 178-193, 200-227,249-258, 262-274, 279-291, 302-308, 322-327, 329-336, 351-363, 366-373,384-399, 403-411, 415-434, 440-446, 461-482, 488-506, 510-516, 518-551,574-589, 607-629, 634-665, 667-687, 694-712, 725-739, 743-751, 753-768KPORF- Zinc-containing 4-13, 19-44, 55-63, 3 G 11-76 25, 09 alcohol71-82, 89-110, 120-130, 212 dehydrogenase 132-138, 145-161, 168-182,189-258, 261-272, 278-288, 290-301 KPORF- Putative inner 4-22, 43-56,63-68, 2 D 383-428 26, 10 membrane protein 81-90, 93-99, 139-148, 213155-160, 170-176, 189-195, 207-218, 227-232, 241-249, 251-258, 260-266,277-295, 300-327, 329-336, 340-356, 384-390, 418-423, 427-433, 438-444KPORF- Putative nucleotide- 10-18, 32-37, 45-55, 8 E 71-85 27, 11utilizing enzyme 60-69, 77-83, 214 related to 89-95, 120-125,molybdopterin- 133-170, 172-185, biosynthesis 193-211, 214-223, 232-249,255-275, 277-303, 305-310, 320-328, 334-341, 347-353, 355-369, 380-386,389-395 KPORF- ABC transporter 4-23, 27-35, 67-73, 2 D  39-110 28, 12ATP-binding protein 80-103, 117-126, 215 132-138, 140-159, 162-171,180-194, 198-208, 211-218, 228-234, 239-253, 262-270, 272-291, 296-305KPORF- Periplasmic 13-24, 27-34, 37-66, 4 D   56-111, 29, 13glycerophosphodiester 69-88, 99-104, 199-261 216 phosphodiesterase149-155, 164-175, 184-193, 199-209, 227-235, 264-273, 276-285, 288-315,323-335, 346-353 KPORF- Glucosamine- 11-22, 25-48, 51-60, 2 G 109-15330, 14 fructose-6-phosphate 64-72, 80-96, 217 aminotransferase 108-122,132-137, 142-150, 152-167, 175-199, 214-229, 237-244, 252-258, 260-266,279-287, 301-340, 345-350 KPORF- Isoleucine tRNA 37-43, 50-57, 65-82, 3G  14-76, 31, 15 synthetase 87-109, 123-129, 176-220 218 141-150,152-157, 166-172, 179-203, 209-241, 249-284, 290-300, 308-326, 329-335,345-357, 359-368, 379-386, 390-417, 420-425, 438-444, 461-466, 473-490,497-505, 524-534, 541-550, 586-597, 608-614, 622-632, 660-666, 679-694,696-706, 708-722, 725-731, 737-763, 784-789, 810-825, 837-854, 857-880,882-895, 901-907, 911-928 KPORF- Putative 9-16, 38-52, 61-86, 5 H113-193 32, 16 oxidoreductase 93-100, 110-117, 219 123-132, 138-145,151-169, 172-181, 186-202, 208-225, 227-253, 264-275, 289-295, 320-329,335-342 KPORF- Putative 11-18, 24-30, 42-49, 2 H 490-547 33, 17molybdopterin 53-63, 69-80, 220 oxidoreductases 87-93, 95-103, 144-171,173-185, 193-200, 202-208, 215-221, 242-261, 266-273, 277-286, 290-299,322-328, 338-351, 354-377, 391-409, 441-451, 461-466, 499-515, 521-527,562-569, 621-629, 647-663, 676-682, 694-701, 703-713, 725-731, 735-744,755-764, 793-800 KPORF- Putative penicillin- 4-11, 14-22, 38-70, 4 C417-489 34, 18 binding protein 81-90, 97-114, 118-132, 221 147-171,173-181, 187-202, 244-250, 252-298, 301-311, 313-331, 342-368, 410-418,446-451, 456-462, 468-474, 476-492, 499-507, 519-528, 552-565, 568-575,584-613, 618-624, 626-649 KPORF- Putative secretion 4-9, 32-53, 66-72, 2G 201-255 35, 19 protein (Multidrug 74-90, 97-104, 110-130, 222resistance efflux 133-139, 144-152, pump) 166-177, 203-213, 215-241,256-275, 291-304, 307-316, 321-326, 334-345, 352-367 KPORF- Hypotheticalprotein, 13-19, 26-43, 66-72, 7 C  25-107 36, 20 no homology 80-85,95-101, 223 109-125, 131-137 KPORF- Regulator, OraA 13-24, 35-43, 50-56,4 D 19-66 37, 21 protein 58-68, 77-83, 224 104-110, 117-125, 132-138,140-153 KPORF- DNA mismatch 15-31, 37-42, 47-54, 2 G 576-623 38, 22repair protein mutS 68-87, 89-96, 225 107-117, 121-127, 131-137,145-151, 176-182, 220-226, 232-246, 250-257, 291-300, 317-325, 328-333,337-359, 368-393, 403-428, 460-478, 480-493, 500-506, 511-516, 519-526,528-559, 565-572, 584-595, 597-605, 608-613, 626-648, 679-684, 687-693,703-714, 718-735, 742-750, 757-765, 768-788, 793-799, 813-819, 823-829,839-850 KPORF- Hydrogenase 10-35, 37-60, 63-76, 2 C 275-339 39, 23isoenzyme HypD 79-86, 88-97, 226 108-113, 118-126, 128-134, 138-145,153-159, 168-188, 194-208, 211-243, 255-260, 270-276, 285-301, 307-346,348-367 KPORF- Selenocysteine lyase 4-17, 21-33, 35-42, 2 H 32-85 40, 2447-64, 72-80, 85-92, 227 98-103, 125-147, 151-161, 165-177, 183-230,232-246, 256-262, 284-306, 310-328, 331-367, 369-383, 392-399 KPORF-Ribose-phosphate 5-11, 18-27, 42-52, 2 H 267-313 41, 25pyrophosphokinase 60-65, 75-84, 90-102, 228 107-116, 125-178, 184-206,221-233, 235-242, 249-257, 264-277, 288-317 KPORF- TonB 5-11, 14-42,50-75, 8 F  85-114 42, 26 79-86, 89-98, 120-125, 229 152-160, 166-181,185-193, 200-207 KPORF- Putative phosphatase 4-30, 36-43, 46-55, 2 D 37-109 43, 27 63-111, 144-152, 230 159-168, 179-189, 191-200, 205-213KPORF- Similarity to 20-45, 57-77, 80-100, 7 A 266-296 44, 28flavoprotein 119-126, 131-137, 231 143-169, 179-185, 195-203, 207-231,235-264, 282-302, 320-329, 341-347, 353-359, 361-373 KPORF- Outermembrane 5-22, 24-37, 41-55, 2 H 103-152 45, 29 channel protein 57-65,72-78, 90-103, 232 105-116, 119-130, 164-170, 190-202, 209-231, 244-254,260-276, 300-339, 344-350, 355-376, 389-397, 399-406, 408-421, 429-437KPORF- Membrane-bound 8-16, 18-25, 31-47, 3 H 405-468 46, 30 ATPsynthase, F1 71-82, 87-102, 104-114, 233 sector, alpha-subunit 126-156,176-183, 190-200, 205-212, 218-228, 231-243, 256-279, 287-301, 303-312,324-332, 335-348, 351-357, 365-380, 395-412, 422-451, 456-464, 467-483,501-507 KPORF- Hypothetical protein 4-18, 21-39, 46-56, 2 C 317-401 47,31 63-69, 72-86, 116-130, 234 132-160, 162-190, 196-201, 209-231,233-241, 251-265, 269-282, 292-298, 309-324, 333-369, 391-415, 417-427,436-454, 471-480, 482-499, 510-518, 521-533, 537-543, 545-561, 571-581,585-597, 599-607, 609-635, 638-643, 650-665, 671-685, 687-695, 701-707,710-720, 724-736, 747-757, 764-769, 772-784, 791-796, 808-820 KPORF-Putative periplasmic 4-12, 15-33, 58-77, 2 D 159-218 48, 32 protein82-89, 98-106, 108-118, 235 120-135, 141-147, 152-160, 168-215, 225-233,235-247, 250-264, 284-312, 314-321, 336-343, 359-374, 386-394 KPORF-D-ribulose-5- 4-16, 24-36, 40-47, 5 H 126-203 49, 33 phosphate 3- 49-56,61-81, 84-143, 236 epimerase 148-156, 158-164, 170-175, 194-206, 208-214KPORF- Hypothetical protein 28-45, 50-61, 94-111, 2 D 549-630 50, 34113-124, 137-142, 237 147-173, 180-188, 190-196, 202-223, 229-235,239-249, 262-270, 280-288, 290-321, 325-332, 347-355, 359-368, 389-407,415-427, 429-453, 458-465, 477-485, 499-505, 516-527, 531-549, 569-592,594-602, 605-615, 628-635, 647-659, 662-683, 727-735, 760-765, 771-780,788-809, 811-818 KPORF- Hypothetical protein 21-28, 33-40, 48-100, 2 H 1-46 51, 35 104-111, 113-134 238 KPORF- 30S ribosomal 12-24, 31-41,53-61, 2 H 26-98 52, 36 subunit protein S5 73-87, 112-128, 239 133-140,151-156 KPORF- Nitrite reductase 4-9, 19-26, 32-56, 2 D 790-834 53, 37large subunit 58-67, 71-81, 90-95, 240 97-105, 112-118, 124-132,138-144, 147-167, 169-177, 199-207, 212-217, 231-241, 250-260, 266-272,274-282, 289-296, 299-310, 316-331, 344-350, 352-363, 368-377, 381-394,399-406, 412-450, 459-473, 486-503, 508-514, 518-548, 564-570, 579-587,602-608, 616-623, 628-635, 638-654, 678-688, 691-696, 703-709, 716-723,761-772, 784-793, 819-826, 835-844 KPORF- Putative inner 4-10, 18-36,43-50, 2 A 761-781 54, 38 membrane 63-71, 75-105, 109-117, 241lipoprotein 134-140, 145-157, 176-182, 184-201, 203-211, 215-225,240-250, 262-284, 294-309, 313-319, 327-337, 350-356, 361-367, 372-393,411-421, 428-451, 453-466, 487-492, 501-528, 535-553, 564-574, 592-605,612-629, 631-640, 646-653, 658-666, 673-681, 713-718, 720-730, 739-749,784-792, 821-826, 833-844, 853-863, 871-876, 885-894, 900-918, 937-950,952-957, 972-990, 995-1001, 1024-1036, 1039-1044, 1049-1055, 1062-1089,1091-1103, 1110-1121, 1123-1129, 1131-1151, 1157-1179, 1181-1201,1204-1223, 1233-1244, 1269-1276, 1279-1286, 1294-1301, 1303-1309,1315-1338, 1350-1362, 1373-1381, 1398-1406, 1412-1423, 1440-1446,1458-1466, 1481-1487, 1492-1508, 1511-1518, 1528-1534, 1536-1547,1553-1565, 1606-1617, 1619-1644 KPORF- Recombinational 6-13, 31-38,47-60, 2 H 161-232 55, 39 DNA repair protein 71-102, 107-123, 242128-155, 173-179, 185-194, 210-220 KPORF- Putative regulatory 11-34,36-43, 49-67, 2 G 130-185 56, 40 protein 74-79, 84-92, 243 94-100,103-112, 120-129, 134-155, 162-173, 177-185, 189-202, 206-211 KPORF- S-4-10, 20-35, 37-46, 4 H 117-201 57, 41 adenosylmethionine 48-55, 60-66,75-82, 244 synthetase 87-98, 133-150, 166-172, 178-189, 208-214,230-235, 245-251, 271-308, 319-333, 335-355, 373-380 KPORF- A/G-specificDNA 4-30, 54-65, 91-105, 1 G 249-323 58, 42 glycosylase 107-131,135-154, 245 163-192, 199-208, 210-224, 229-239, 248-257, 263-279,281-294, 328-354, 373-379, 382-405, 426-453, 462-487 KPORF- Hypotheticalprotein 4-10, 12-24, 45-55, 6 E 24-40 59, 43 75-88 246 KPORF-Transketolase 4-14, 20-37, 47-53, 2 D 232-278 60, 44 55-61, 75-81,97-103, 247 107-124, 129-135, 139-147, 160-166, 169-175, 181-190,202-221, 247-255, 272-285, 300-310, 318-332, 351-361, 384-397, 406-427,442-449, 458-482, 494-503, 512-524, 531-539, 552-562, 577-588, 590-596,600-608, 613-624, 637-668, 692-700 KPORF- Putative S- 33-39, 49-55,68-84, 4 G, H   63-115, 61, 45 adenosylmethionine- 90-96, 104-120,200-250 248 dependent 126-143, 150-159, methyltransferase 168-191,197-208, 219-225, 227-233, 241-247 KPORF- Nucleoside- 4-22, 24-34,36-55, 5 G, H 117-183 62, 46 diphosphate-sugar 57-76, 83-97, 99-117, 249epimerase 135-143, 145-157, 163-174, 178-198, 200-207, 209-270, 276-290,321-335, 338-347, 367-374, 393-402, 404-411, 416-422, 443-460, 467-473KPORF- Coenzyme F420- 26-37, 44-52, 57-96, 6 G  47-105 63, 47 dependentN5,N10- 104-111, 118-124, 250 methylene 155-177, 179-197,tetrahydromethanopterin 201-214, 223-233, reductase and 243-250,257-262, related flavin- 291-297, 303-314, dependent 319-363oxidoreductases KPORF- ATP/GTP-binding 36-43, 45-60, 76-97, 2 G 118-16364, 48 protein 107-125, 131-156, 251 158-164 KPORF- ABC transporter,5-32, 40-50, 52-60, 3 D 209-255 65, 49 substrate binding 70-88, 92-101,106-126, 252 protein 138-150, 152-161, 175-193, 201-234, 237-248,270-285, 297-303, 312-318 KPORF- Putative PTS system 4-12, 23-34, 49-55,2 D  62-113 66, 50 IIA component 59-65, 70-81, 83-130 253 KPORF-Putative phosphatase 4-26, 38-49, 69-76, 3 D 100-167 67, 51 82-96,103-119, 254 126-140, 143-190, 194-209, 212-218 KPORF- Putativelipoprotein 7-29, 35-47, 56-66, 18 C  75-176 68, 52 80-94, 97-123,125-148, 255 150-160, 166-173, 175-191, 193-200, 207-225 KPORF-Conserved 14-36, 39-45, 51-59, 9 G 202-256 69, 53 hypothetical protein66-71, 76-88, 256 106-117, 121-126, 140-157, 164-187, 198-206, 210-252KPORF- Membrane-bound 4-19, 27-35, 90-107, 2 D 130-178 70, 54 lyticmurein 120-134, 144-150, 257 transglycosylase d 166-175, 192-198,precursor 221-243, 249-255, 263-278, 283-288, 305-321, 324-334, 342-349,355-366, 377-390, 413-425, 442-448 KPORF- Similar to rRNA 17-26, 41-51,54-61, 4 D  69-126 71, 55 methylases 64-72, 78-105, 258 117-125,127-137, 147-155, 175-213, 230-236, 238-261, 271-277, 282-297, 309-318,329-347, 355-372, 377-390 KPORF- Apolipoprotein N- 4-48, 54-60, 62-69, 2G 226-282 72, 56 acyltransferase 73-81, 88-115, 124-137, 259 139-154,156-169, 171-190, 194-231, 240-273, 288-303, 336-363, 367-395, 405-411,434-442, 449-454, 466-483, 491-507 KPORF- Putative carboxylase 26-34,39-47, 50-80, 2 D 149-239 73, 57 82-88, 97-105, 260 108-127, 131-137,162-180, 185-191, 198-203, 209-214, 226-247, 256-288, 296-305 KPORF-Putative transport 5-28, 30-54, 73-84, 2 D  6-62 74, 58 protein 89-98,109-116, 261 122-128, 137-142, 163-189, 207-236, 245-280, 288-390,404-423, 426-433, 450-474, 487-504, 506-513, 524-530, 532-595, 605-614,620-626, 631-638, 644-657, 667-683, 686-693, 695-702, 707-733, 739-747KPORF- Similarity Anaerobic 23-31, 39-50, 55-67, 4 A 612-626 75, 59dehydrogenases 76-100, 117-130, 262 149-171, 173-185, 218-238, 242-288,291-298, 334-346, 355-369, 382-399, 413-420, 431-438, 442-449, 455-466,486-493, 498-508, 524-531, 540-546, 551-558, 562-570, 575-582, 585-596,598-604, 621-630, 632-650, 670-677, 682-701, 736-749, 755-761 KPORF-Similarity glutamine- 4-21, 24-39, 44-68, 2 A 126-148 76, 60 bindingperiplasmic 74-81, 85-91, 109-116, 263 protein precursor 129-138,142-148, 173-188, 195-201, 207-212, 223-228 KPORF- Putative cytoplasmic4-17, 24-42, 61-67, 7 G, H  1-49 77, 61 protein 84-93, 96-102, 116-121,264 135-143, 155-165, 177-186, 210-224, 253-259, 272-297, 299-331,337-351, 359-367, 369-385 KPORF- Probable 4-25, 28-54, 67-81, 4 G127-182 78, 62 transcriptional 85-136, 138-143, 265 regulator 157-170,180-190, 197-203, 205-214, 219-243, 246-270, 277-283, 290-299, 305-311KPORF- Hypothetical protein 11-20, 25-33, 75-80, 2 D 128-176 79, 6385-91, 113-124, 266 143-155, 161-170, 172-184 KPORF- Extracellularsolute- 4-9, 16-26, 28-34, 26 G  48-106 80, 64 binding protein, 55-80,120-143, 267 family 3 150-156, 158-164, 167-178, 185-190, 192-213,221-237, 242-255, 257-272, 281-290, 325-332 KPORF- Penicillin-binding13-48, 59-70, 78-88, 4 C 410-466 81, 65 protein 95-112, 129-151, 268153-161, 163-182, 214-221, 235-245, 248-277, 281-291, 293-301, 303-311,315-320, 323-346, 377-383, 390-398, 447-454, 474-487, 491-512, 531-544,547-553, 582-590, 597-603, 605-611, 623-629 KPORF- Outer membrane 6-26,39-46, 48-58, 2 D 152-206 82, 66 porin, receptor for 69-75, 109-121, 269ferric enterobactin 139-144, 148-155, (enterochelin) and 166-172,215-221, colicins B and D 261-267, 313-319, 363-386, 423-433, 447-458,465-471, 483-494, 497-517, 558-565, 578-586, 589-597, 619-626, 636-645,659-665, 671-680, 682-693, 733-739 KPORF- Hypothetical protein 4-19,23-35, 40-50, 2 H 29-90 83, 67 52-58, 65-73, 78-103, 270 112-125,146-160, 163-192, 194-200 KPORF- Hypothetical protein 4-13, 17-32,40-50, 2 D  90-170 84, 68 57-67, 76-81, 88-95, 271 107-119, 131-142,144-157, 171-178, 185-193, 197-207, 212-227, 231-238, 248-253, 263-310KPORF- 3-phytase precursor 9-28, 57-82, 84-93, 2 D  80-141 85, 69126-135, 143-166, 272 173-194, 196-201, 212-220, 228-254, 269-277,289-298, 305-316, 320-327, 330-337, 350-359, 373-378, 386-392, 403-411,421-428, 435-441, 443-458, 465-470 KPORF- Cation/multidrug 11-48, 54-67,69-75, 1 H 775-825 86, 70 efflux pump 89-95, 101-122, 273 124-131,134-157, 159-175, 202-208, 214-228, 258-270, 272-280, 287-295, 298-310,331-338, 340-417, 427-500, 502-509, 534-552, 556-561, 564-577, 585-592,594-608, 621-627, 632-641, 643-652, 671-681, 683-709, 712-743, 758-764,776-783, 789-820, 835-851, 864-883, 885-910, 913-940, 948-953, 967-976,994-1020 KPORF- ferric enterobactin 14-24, 32-54, 58-63, 5 H 10-59 87,71 transport ATP- 70-80, 93-100, 274 binding protein 108-125, 127-135,142-153, 155-160, 180-191, 201-208, 210-216, 222-235, 242-264, 267-273,276-282, 284-308 KPORF- ATP-dependent 16-28, 44-68, 70-77, 2 D 555-62188, 72 protease La 83-90, 99-129, 275 131-137, 145-154, 161-175,183-190, 196-203, 205-220, 238-245, 321-328, 330-338, 366-379, 383-397,399-405, 412-418, 442-458, 471-483, 486-505, 536-544, 562-568, 583-602,610-618, 629-635, 641-655, 672-682, 697-705, 714-729, 744-751, 755-762,766-771, 783-807 KPORF- Predicted Fe—S 4-9, 20-34, 45-54, 2 D 100-14089, 73 oxidoreductase 60-77, 79-89, 91-100, 276 102-149, 162-170,177-189, 193-208, 210-222, 238-244, 252-264, 267-276, 302-307 KPORF-Probable 11-27, 30-49, 56-62, 10 G, H  73-137 90, 74 transcriptional69-74, 76-85, 277 regulator, LysR 94-108, 116-125, family 129-147,153-161, 165-171, 177-208, 217-223, 225-231, 237-255, 260-284, 293-300KPORF- Hypothetical protein 4-38, 40-51, 84-97, 2 E 727-740 91, 7599-106, 109-115, 278 119-129, 131-145, 148-160, 180-186, 188-202,230-243, 246-267, 274-288, 290-299, 302-312, 317-327, 332-344, 353-377,381-388, 407-419, 423-437, 447-470, 474-482, 486-494, 501-523, 531-546,551-556 KPORF- Hypothetical protein 23-52, 62-76, 87-104, 3 C 272-32492, 76 109-115, 117-123, 279 129-139, 143-149, 152-170, 172-191,199-205, 212-218, 220-240, 249-256, 263-275, 297-303, 308-342, 349-380,382-394, 414-420, 430-441, 446-452, 460-475, 488-505, 514-531, 533-539,546-568, 570-577, 579-588, 613-625, 632-670, 672-716, 718-745, 759-769,785-798, 801-807 KPORF- Hypothetical protein, 4-34, 36-43, 56-73, 2 C241-296 93, 77 (located as CRF in 80-87, 101-134, 280 ABC transporter148-159, 161-170, substrate-binding 178-185, 195-206, protein) 211-221,223-248, 259-271, 276-295, 297-308 KPORF- Maltoporin 5-31, 44-50, 64-74,4 E 166-202 94, 78 86-94, 132-147, 281 154-167, 196-203, 209-219,253-260, 284-289, 300-312, 319-327, 335-340, 358-364, 376-383 KPORF-Hypothetical protein 4-9, 12-27, 29-71, 3 G 317-364 95, 79 77-84,90-108, 114-142, 282 147-164, 180-213, 217-227, 229-282, 291-309,322-329, 336-353, 365-370 KPORF- Methyl-accepting 36-41, 52-66, 71-83, 2H  1-50 96, 80 chemotaxis protein 89-95, 116-127, 283 154-174, 176-184,200-206, 230-237, 248-259, 269-284, 307-316, 376-383, 399-418, 424-442,445-451, 454-462 KPORF- Type I restriction- 9-14, 33-49, 64-72, 4 C 212-244, 97, 81 modification system 87-92, 103-109, 533-611 284 DNAmethylase 123-128, 130-141, 143-154, 160-166, 182-214, 237-247, 251-260,292-300, 327-332, 337-350, 357-365, 388-398, 405-411, 422-428, 451-459,478-488, 520-531, 534-540, 558-564, 580-586, 591-600, 605-615, 629-635,641-653, 658-672 KPORF- Pyruvate 4-10, 17-27, 30-37, 2 C 277-324 98, 82dehydrogenase E2 44-62, 80-85, 94-114, 285 component 118-131, 134-141,148-161, 171-212, 218-241, 248-261, 274-313, 325-336, 342-348, 359-373,391-397, 424-431, 454-474, 489-495, 497-503, 505-515, 548-553, 560-580,591-610 KPORF- Sulfite reductase 7-16, 18-24, 30-47, 23 H 335-389 99, 83[NADPH] 49-70, 83-99, 103-117, 286 flavoprotein alpha- 126-141, 146-153,component 159-165, 177-194, 198-221, 236-246, 255-262, 273-279, 283-296,301-332, 338-411, 422-428, 434-440, 452-458, 463-469, 494-509, 511-517,524-531, 548-554, 564-572 KPARF- Hypothetical protein 9-15, 33-54,56-80, 10 G  1-42 100, 01 102-108 287 KPARF- Hypothetical protein 15-36,42-55, 58-68 83 E, F 54-77 101, 02 288 KPARF- Hypothetical protein55-75, 89-96, 98-110 51 E, F 14-36 102, 03 289 KPARF- Hypotheticalprotein 8-14, 29-51, 73-101, 31 E, H  70-114 103, 04 110-117 290 KPARF-Hypothetical protein 20-25, 29-34, 41-52, 5 H 21-58 104, 05 60-67,69-85, 291 90-100, 114-122, 136-142, 160-170, 174-181 KPARF-Hypothetical protein 14-22 33 E, F  4-13 105, 06 292 KPARF- Hypotheticalprotein 22-40, 54-66, 88-105, 4 G 31-74 106, 07 109-118 293 KPARF-Hypothetical protein 5-11, 18-32, 47-60, 14 F 118-129 107, 08 66-73,83-92, 113-120, 294 126-141, 151-164, 167-174, 201-211 KPARF-Hypothetical protein 5-11, 18-24, 32-40, 38 E 25-54 108, 09 47-53 295KPARF- Hypothetical protein 18-24, 31-48 22 H  5-55 109, 10 296 KPARF-Hypothetical protein 10-16, 26-32, 47-56, 9 G 10-62 110, 11 85-95 297KPARF- Hypothetical protein 4-12, 16-26 9 F 25-34 111, 12 298 KPARF-Hypothetical protein 19-29, 45-51, 63-68, 3 H 14-78 112, 13 76-92,103-110, 299 114-120, 123-133, 135-141 KPARF- Hypothetical protein 4-18,47-61 3 E 57-93 113, 14 300 KPARF- Hypothetical protein 17-29, 44-50 5 F26-38 114, 15 301 KPARF- Hypothetical protein 5-19, 55-64, 78-85, 2 A24-33 115, 16 95-101, 104-112 302 KPARF- Hypothetical protein 4-10 3 A12-31 116, 17 303 KPARF- Hypothetical protein 4-12, 27-41, 43-58, 3 G13-65 117, 18 60-67, 76-86 304 KPARF- Hypothetical protein 30-38, 57-672 F  5-32 118, 19 305 KPARF- Hypothetical protein 30-43 4 A  2-21 119,20 306 KPARF- Hypothetical protein 14-20, 23-36, 41-48 3 C  1-52 120, 21307 KPARF- Hypothetical protein 18-33, 51-58, 76-82 3 B 32-46 121, 22308 KPARF- Hypothetical protein 25-31 2 E  2-16 122, 23 309 KPARF-Hypothetical protein 14-23, 50-58 3 G  9-49 123, 24 310 KPARF-Hypothetical protein 4-10, 22-31, 35-45, 2 G 17-66 124, 25 48-68, 71-80311 KPARF- Hypothetical protein 4-24, 28-42, 46-56, 3 H  2-46 125, 2663-69, 87-94, 112-131 312 KPARF- Hypothetical protein 4-15, 19-28,34-41, 3 E  2-20 126, 27 52-62, 78-86 313 KPARF- Hypothetical protein4-11, 16-30, 32-42 5 H  7-38 127, 28 314 KPARF- Hypothetical protein4-20, 22-31 7 A 22-38 128, 29 315 KPARF- Hypothetical protein 4-19 3 F17-32 129, 30 316 KPARF- Hypothetical protein 7-13, 17-22, 27-33, 2 F26-40 130, 31 80-100 317 KPARF- Hypothetical protein 10-18, 22-48 2 E32-44 131, 32 318 KPARF- Hypothetical protein 15-24, 43-49, 73-83 2 G45-93 132, 33 319 KPARF- Hypothetical protein 22-29, 46-55, 57-63 3 A 5-17 133, 34 320 KPARF- Hypothetical protein 10-33 2 F 21-35 134, 35321 KPARF- Hypothetical protein 16-24 2 A 22-49 135, 36 322 KPARF-Hypothetical protein 4-16, 37-73, 76-110, 2 A  2-30 136, 37 117-125,127-132 323 KPARF- Hypothetical protein 4-12, 23-35, 44-56, 2 H 22-76137, 38 59-88 324 KPARF- Hypothetical protein 15-26 2 F 23-35 138, 39325 KPARF- Hypothetical protein 12-22, 31-40 2 F 17-44 139, 40 326KPARF- Hypothetical protein 4-9, 13-18, 29-35 2 B 57-64 140, 41 327KPARF- Hypothetical protein 31-55, 67-81 2 H 25-70 141, 42 328 KPARF-Hypothetical protein 13-24, 51-58 2 E 13-26 142, 43 329 KPARF-Hypothetical protein 6-20, 29-40, 57-79 3 H 46-88 143, 44 330 KPARF-Hypothetical protein 8-14, 41-54, 68-76, 3 G 12-72 144, 45 83-93,106-126, 331 130-139 KPARF- Hypothetical protein 5-13, 17-24, 41-55, 5 G53-88 145, 46 64-69, 80-85, 94-107, 332 109-115 KPARF- Hypotheticalprotein 5-12, 32-54, 57-64 2 A 20-33 146, 47 333 KPCRF- Hypotheticalprotein 4-16, 40-48, 50-58, 5 C  68-128 147, 01 62-68, 75-85, 92-104,334 108-116, 124-134 KPCRF- Hypothetical protein 7-13, 19-29, 34-40, 19D, F 11-83 148, 02 54-71, 76-81, 91-144, 335 147-155, 157-188 KPCRF-Hypothetical protein 17-24, 32-41 79 A, E, F  6-43 149, 03 336 KPCRF-Hypothetical protein 14-31, 38-59, 69-87, 11 E 63-78 150, 04 95-102,126-146, 337 157-162, 177-193, 201-227, 238-251 KPCRF- Hypotheticalprotein 10-16, 18-25, 27-41, 5 H  38-100 151, 05 43-52, 59-86, 33894-101, 134-140 KPCRF- Hypothetical protein 4-19, 23-35, 43-72, 3 H37-93 152, 06 78-92 339 KPCRF- Hypothetical protein 15-20, 27-32, 41-65,2 C 317-375 153, 07 69-82, 93-105, 340 107-115, 120-147, 170-178,184-201, 214-257, 272-281, 293-314, 332-339, 358-364, 374-381, 390-397,399-414, 428-460 KPCRF- Hypothetical protein 11-28, 47-55, 59-68, 2 A144-158 154, 08 76-105, 108-116, 341 120-144, 146-160, 167-175, 180-187,209-233 KPCRF- Hypothetical protein 4-13, 58-78 31 H 14-77 155, 09 342KPCRF- Hypothetical protein 26-31, 44-49, 57-64, 30 A, E, F, G  57-101156, 10 67-74, 107-112, 343 116-152, 154-181, 202-212, 241-255 KPCRF-Hypothetical protein 10-41, 53-70, 81-93, 21 E, F 84-95 157, 11 100-111,137-147, 344 164-169, 183-190, 199-210, 216-221, 226-240 KPCRF-Hypothetical protein 12-45, 48-56, 73-79, 3 F  98-115 158, 12 91-103,106-112, 345 117-125, 132-143, 154-160, 178-201, 208-214, 216-225,260-266, 276-283 KPCRF- Hypothetical protein 4-15, 30-42 2 B 29-39 159,13 346 KPCRF- Hypothetical protein 22-53, 55-73, 80-88 3 A 33-66 160, 14347 KPCRF- Hypothetical protein 6-23, 44-54 7 F 56-67 161, 15 348 KPCRF-Hypothetical protein 8-21, 35-44, 66-75, 3 C 32-94 162, 16 82-87, 94-101349 KPCRF- Hypothetical protein 8-20, 23-32, 36-50, 4 H 15-69 163, 1753-69 350 KPCRF- Hypothetical protein 5 F  8-22 164, 18 351 KPCRF-Hypothetical protein 31-37 5 A  2-31 165, 19 352 KPCRF- Hypotheticalprotein 4-20, 23-39, 58-63, 2 D 22-82 166, 20 71-78, 97-102 353 KPCRF-Hypothetical protein 23-44, 135-152, 2 C  57-116 167, 21 168-184 354KPCRF- Hypothetical protein 24-31, 42-50, 52-62, 2 D 43-94 168, 2293-117 355 KPCRF- Hypothetical protein 20-29 10 E 24-43 169, 23 356KPCRF- Hypothetical protein 12-57, 59-74 2 A 22-40 170, 24 357 KPCRF-Hypothetical protein 7-16, 18-26, 39-45, 2 A 65-82 171, 25 68-78, 86-92358 KPCRF- Hypothetical protein 5-17, 19-34, 42-48, 6 H  67-111 172, 2656-71, 102-113, 359 118-129 KPCRF- Hypothetical protein 4-33, 50-71 3 D13-55 173, 27 360 KPCRF- Hypothetical protein 9-17, 23-30, 37-54, 2 D 5-70 174, 28 69-88, 96-102, 114-123, 361 130-140, 143-163 KPCRF-Hypothetical protein 4-23, 27-52, 71-80 11 C, H  9-94 175, 29 362 KPCRF-Hypothetical protein 13-19 5 A  2-21 176, 30 363 KPCRF- Hypotheticalprotein 18-26, 28-52, 63-74, 2 D 18-84 177, 31 94-107, 123-134 364KPCRF- Hypothetical protein 19-33, 57-68 3 A 26-48 178, 32 365 KPCRF-Hypothetical protein 4-26, 31-37, 42-59 7 H 12-65 179, 33 366 KPCRF-Hypothetical protein 4-25 1 A 20-39 180, 34 367 KPCRF- Hypotheticalprotein 40-51, 54-62, 67-75, 3 A 31-42 181, 35 83-89, 126-146, 368148-156 KPCRF- Hypothetical protein 4-15, 23-33, 38-49, 2 H  7-91 182,36 82-98 369 KPCRF- Hypothetical protein 6-26, 36-57 2 F 40-64 183, 37370 KPCRF- Hypothetical protein 6-15, 21-28, 32-38, 13 H 41-95 184, 3857-65, 78-103, 114-134, 371 138-144, 154-163 KPCRF- Hypothetical protein13-30, 47-57, 71-76 2 C 25-71 185, 39 372 KPCRF- Hypothetical protein4-31, 43-51, 55-63, 2 F  82-118 186, 40 67-72, 76-83, 88-95, 373 99-118,125-132, 134-159 KPCRF- Hypothetical protein 4-17, 26-32, 34-40, 2 H41-97 187, 41 45-61, 67-92 374 A, 50 bp library of K. pneumoniae in lamBwith the ICKp18-IgG pool containing the IC38, IC40, IC76 and IC86 (562trimmed clones), B, 50 bp library of K. pneumoniae in lamB with theICKp19-IgG pool containing the IC88, IC89, IC92 and IC93 sera (444trimmed clones), C, 300 bp library of K. pneumoniae in fhuA with theICKp18-IgG pool containing the IC38, IC40, IC76 and IC86 sera (455trimmed clones), D, 300 bp library of K. pneumoniae in fhuA with theICKp19-IgG pool containing the IC88, IC89, IC92, and IC93 sera (591trimmed clones), E, 50 bp library of K. pneumoniae in lamB with thePKp34 pool (P3536.2, P3548, P3560, P3582 and P3583 sera) (618 trimmedclones), F, 50 bp library of K. pneumoniae in lamB with the PKp35 pool(P3495.2, P3533.2, P3567 and P3576 sera) (562 trimmed clones), G, 300 bplibrary of K. pneumoniae in fhuA with the PKp34 pool (P3536.2, P3548,P3560, P3582 and P3583 sera) (562 trimmed clones), H, 300 bp library ofK. pneumoniae in fhuA with the PKp35 pool (P3495.2, P3533.2, P3567 andP3576 sera) (593 trimmed clones); P3536.2, P3495.2 and P3533.2 wereconvalescent sera obtained from patients after recovering from sickness,*prediction of antigenic sequences longer than 5 amino acids wasperformed with the program ANTIGENIC (Kolaskar, A. et al., 1990). Listedare the genes from K. pneumoniae as identified by BLAST of thedetermined epitope sequence against the genomic sequence of K.pneumoniae MGH78578 (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi,http://pedant.gsf.de/). The numbering of the ORFs is arbitrary. Theannotation/putative function was mainly obtained by homology to openreading frames from other bacterial species, preferentially Gramnegative bacteria.

TABLE 2 List of strains used for gene distribution analysis. No StrainID Species K-type O-type 1 A 5054 K. pneumoniae 1 1 2 i252/94 K.pneumoniae 1 3 3 B 5055 K. pneumoniae 2 1 4 i225/94 K. pneumoniae 3 1 5C 5046 K. pneumoniae 3 2 6 D 5050 K. ozaenae 4 2ac 7 E 5051 K. ozaenae 52ac 8 Aerogenes 4140 K. pneumoniae 7 1 9 1015 K. planticola 8 1 10i272/94 K. pneumoniae 8 3 11 919 K. pneumoniae 10 1 12 313 K. pneumoniae12 1 13 1470 K. pneumoniae 13 O− 14 1193 K. planticola 14 5 15 Mich61 K.pneumoniae 15 4 16 2069/49 K. pneumoniae 16 1 17 i243/94 K. pneumoniae16 3 18 1702/49 K. pneumoniae 21 1 19 i202/94 K. pneumoniae 21 3 201680/49 K. pneumoniae 24 1 21 i257/94 K. pneumoniae 24 9 22 2002/49 K.pneumoniae 25 3 23 6613 K. pneumoniae 27 2 24 i192/94 K. pneumoniae 27 925 5758 K. pneumoniae 28 2 26 5725y K. oxytoca 29 1 27 i219/94 K.pneumoniae 29 3 28 6258 K. pneumoniae 31 3 29 6168 K. pneumoniae 33 3 30i256/94 K. pneumoniae 38 3 31 8414 K. pneumoniae 38 O− 32 7749 K.planticola 39 1 33 5281 K. pneumoniae 46 1 34 i224/94 K. pneumoniae 52 335 5759/50 K. pneumoniae 52 O− 36 Stanley K. planticola 54 3 37 i221/94K. pneumoniae 57 3 38 4425/51 K. planticola 57 5 39 264-1 K. pneumoniae67 7 40 265-1 K. pneumoniae 68 2 41 i203/94 K. pneumoniae 68 3 42 889 K.pneumoniae 69 8 43 708 K. pneumoniae 80 12  44 370 K. pneumoniae 81 O+45 Friedländer 204 K. pneumoniae K− 1 46 5053 K. ozaenae K− 2a, 2c 47MGH78578 K. pneumoniae nd nd Table 2 shows different strains of K.pneumoniae isolates analyzed for the gene distribution study. Thespecies and the relevant K- and O-type are given. MGH78578 was used forgenerating genomic libraries. nd, not determined.

TABLE 3 Gene distribution analysis for a selected number of antigens invarious Klebsiella species and K. pneumoniae strains. SEQ ID NO Gene ORF(DNA) distribution KPORF-01 17 46/46 KPORF-02 18 36/46 KPORF-03 19 12/46KPORF-04 20 42/46 KPORF-05 21 46/46 KPORF-06 22 17/46 KPORF-07 23 41/46KPORF-08 24  5/46 KPORF-09 25 41/46 KPORF-10 26 43/46 KPORF-11 27 46/46KPORF-12 28 22/46 KPORF-13 29 43/46 KPORF-14 30 45/46 KPORF-15 31 40/46KPORF-16 32 32/46 KPORF-17 33 34/46 KPORF-18 34 39/46 KPORF-19 35 24/46KPORF-20 36 39/46 KPORF-21 37 43/46 KPORF-22 38 27/46 KPORF-23 39  4/46KPORF-24 40 28/46 KPORF-25 41 46/46 KPORF-26 42 35/46 KPORF-27 43 43/46KPORF-28 44 41/46 KPORF-29 45 40/46 KPORF-30 46 46/46 KPORF-31 47 46/46KPORF-32 48 46/46 KPORF-33 49 46/46 KPORF-34 50 38/46 KPORF-35 51 39/46KPORF-36 52 46/46 KPORF-37 53 46/46 KPORF-38 54 41/46 KPORF-39 55 39/46KPORF-40 56 38/46 KPORF-41 57 39/46 KPORF-42 58 46/46 KPORF-43 59 37/46KPORF-44 60 45/46 KPORF-45 61 46/46 KPORF-46 62 39/46 KPORF-47 63  1/46KPORF-48 64 39/46 KPORF-49 65 46/46 KPORF-50 66 46/46 KPORF-51 67  7/46KPORF-52 68  7/46 KPORF-53 69 46/46 KPORF-54 70 46/46 KPORF-55 71 46/46KPORF-56 72 44/46 KPORF-57 73 40/46 KPORF-58 74 42/46 KPORF-59 75 17/46KPORF-60 76 46/46 KPORF-61 77 44/46 KPORF-62 78 43/46 KPORF-63 79 35/46KPORF-64 80 40/46 KPORF-65 81 46/46 KPORF-66 82 42/46 KPORF-67 83 18/46KPORF-68 84 37/46 KPORF-69 85  5/46 KPORF-70 86 42/46 KPORF-71 87 39/46KPORF-72 88 43/46 KPORF-73 89 41/46 KPORF-74 90 36/46 KPORF-75 91 31/46KPORF-76 92 38/46 KPORF-77 93 37/46 KPORF-78 94 40/46 KPORF-79 95  4/46KPORF-80 96  4/46 KPORF-81 97  2/46 KPORF-82 98 46/46 KPARF-01 100 37/46KPARF-04 103 40/46 KPARF-05 104 46/46 KPARF-07 106 43/46 KPARF-08 10740/46 KPCRF-01 147 39/46 KPCRF-02 148  1/46 KPCRF-05 151 10/46 KPCRF-06152 45/46 KPCRF-07 153 44/46 KPCRF-08 154 36/46 KPCRF-10 156 32/46KPCRF-12 158 34/46 46 Klebsiella strains plus MGH78578 as a positive PCRcontrol as shown in Table 3 were tested by PCR with oligonucleotidesspecific for the genes encoding relevant antigens. The gene distributiontable lists the number of positive PCR results from 46 strains for eachgene and is an indication of the presence and conservation of the genein diverse isolates of Klebsiella species.

TABLE 4 Peptide ELISA with peptides derived from K. pneumoniae antigensImmune reactivity of individual synthetic peptides representing selectedepitopes with individual human sera is shown. Extent of reactivity iscolour coded; white, < 0.05 OD units; light grey, 0.05- 0.2 OD units;dark grey, 0.2-0.4 OD units; black, > 0.4 OD units. The “Sum” representsthe number of sera, for which the OD_(405 nm) measurement was at least0.05 OD units above the blank without coating. Score is calculated asthe sum of all reactivities (white = 0; light grey = 1; dark grey = 2;black = 3). “From aa” and “To aa” denotes the position of the peptiderelative to the full length protein as listed under the respectivesequence identification number (Seq ID No). ELISA experiments werepreformed with peptides derived from K. pneumoniae antigens and 22 hightiter human sera (P3494.2, P3495.2, P3518.2, P3533.2, P3536.2, P3545,P3548, P3560, P3567, P3571, P3576, P3581, P3582, P3583, IC38, IC40,IC76, IC86, IC88, IC89, IC92 and IC93). P3494.2, P3495.2, P3518.2,P3533.2 and P3536.2 are convalescent sera.

TABLE 5 Surface staining with epitope sera generated in mice. LocationSeq ID in NO protein Friedländer ORF (Protein) (aa) A5054 204 KPORF-02205  46-105 +++ ++ KPORF-13 216  56-111 ++ +++ KPORF-20 223  25-107 ++++ KPORF-21 224 19-66 ++ ++ KPORF-26 229  85-114 + ++ KPORF-27 230 37-109 + ++ KPORF-28 231 266-296 + ++ KPORF-29 232 103-152 + +++KPORF-32 235 167-218 + +++ KPORF-37 240 790-834 + +++ KPORF-38 241761-781 ++ +++ KPORF-39 242 176-232 ++ +++ KPORF-41 244 117-201 + ++KPORF-42 245 249-323 + ++ KPORF-44 247 232-278 + ++ KPORF-49 252209-255 + +++ KPORF-52 255  75-176 + ++ KPORF-53 256 202-256 + ++KPORF-54 257 130-178 + ++ KPORF-55 258  69-126 ++ +++ KPORF-60 263126-148 + ++ KPORF-61 264  1-49 + ++ KPORF-62 265 127-182 + ++ KPORF-64267  48-106 + ++ KPORF-65 268 410-466 + +++ KPORF-66 269 152-206 ++ +++KPORF-72 275 555-621 + ++ KPORF-78 281 166-202 ++ +++ KPORF-79 282317-364 + ++ KPORF-80 283  1-50 + ++ KPORF-82 285 277-324 ++ +++KPARF-03 289 14-36 ++ +++ KPCRF-03 336  6-43 ++ +++ KPCRF-10 343  57-101++ ++ KPCRF-11 344 84-95 +++ +++ KPCRF-12 345  98-115 + +++ The epitopespecific antibodies generated in mice were tested in FACS analysis forbinding to the surface of K. pneumoniae strain A5054 and Friedländer 204cells. The sera that showed a significant shift in FACS analysis arelisted. The extent of the shift is indicated by the number of “+”; +,0-9%; ++, 10-35%, +++, >36%. The percentage indicates the number ofcells that showed a shift in the FACS analysis in comparison to cellsincubated without immune sera.

TABLE 6 Klebsiella sp. strains utilized for the gene conservationanalyses of Klebsiella pneumoniae antigens. Number Strain name SpeciesK-Type O-Type 1 A 5054 K. pneumoniae 1 1 2 i252/94 K. pneumoniae 1 3 3 B5055 K. pneumoniae 2 1 4 i225/94 K. pneumoniae 3 1 5 D 5050 K. ozaenae 42ac 6 E 5051 K. ozaenae 5 2ac 7 Aerogenes4140 K. pneumoniae 7 1 8 1015K. planticola 8 1 9 i262/94 K. pneumoniae 9 3 10 919 K. pneumoniae 10 111 313 K. pneumoniae 12 1 12 1470 K. pneumoniae 13 O− 13 1193 K.planticola 14 5 14 Mich61 K. pneumoniae 15 4 15 2069/49 K. pneumoniae 161 16 i243/94 K. pneumoniae 16 3 17 2005/49 K. pneumoniae 17 O− 181754/49 K. pneumoniae 18 1 19 1702/49 K. pneumoniae 21 1 20 i202/94 K.pneumoniae 21 3 21 1996/49 K. pneumoniae 22 1 22 1680/49 K. pneumoniae24 1 23 2002/49 K. pneumoniae 25 3 24 6613 K. pneumoniae 27 2 25 5758 K.pneumoniae 28 2 26 5725y K. oxytoca 29 1 27 7824 K. pneumoniae 30 1 286258 K. pneumoniae 31 3 29 6168 K. pneumoniae 33 3 30 i256/94 K.pneumoniae 38 3 31 7749 K. planticola 39 1 32 2482 K. pneumoniae 43 2 335281 K. pneumoniae 46 1 34 i224/94 K. pneumoniae 52 3 35 1756/51 K.planticola 53 3 36 Stanley K. planticola 54 3 37 3985/51 K. pneumoniae55 3 38 i221/94 K. pneumoniae 57 3 39 4463/52 K. pneumoniae 60 5 405710/52 K. pneumoniae 61 5 41 5711/52 K. pneumoniae 62 1 42 5845/52 K.pneumoniae 63 1 43 NCTC8172 K. pneumoniae 64 1 44 264-1 K. pneumoniae 677 45 265-1 K. pneumoniae 68 2 46 889 K. pneumoniae 69 8 47 708 K.pneumoniae 80 12  48 370 K. pneumoniae 81 O+ 49 Friedländer 204 K.pneumoniae K− 1 50 5053 K. ozaenae K− 2a, 2c 51 MGH78578 K. pneumoniaend nd

TABLE 7Oligonucleotides used for sequence conservation analyses. Shown arethe KPORFs and primer names, SEQ ID NOs, orientation of the primerrelative to the gene, the sequence, and the position relative to the gene. Oligonucleotides were used for both PCR amplification  of the gene or gene fragment and subsequent sequence analyses. PositionPrimer relative ORF name Orientation Sequence to gene start KPORF-13210-5901 sense GTGCTGGCGGTTATCCTG   −83 to −66 (SEQ ID NO 377) 210-5902antisense GTTATTCCCGGGTCGAAATC +1145 to 1164 (SEQ ID NO 378) KPORF-21210-5903 sense ACGCAGAAGAAACCGAACAG   −89 to −70 (SEQ ID NO 379)210-5904 antisense CGACGGAATAAAGTGGGAAT  +549 to +568 (SEQ ID NO 380)KPORF-32 210-6097 sense GACCAGAGTGAAATATTTACAAAA   −52 to −29(SEQ ID NO 381) 210-6563 antisense GTTTATCGCCACGCTGAT +1295 to +1312(SEQ ID NO 382) KPORF-37 210-5905 sense GCCTGATGGCTGAATCGTTA  −54 to −35 (SEQ ID NO 383) 210-5906 antisense GAGCAGCGTTTTGTTGTCG+1089 to +1107 (SEQ ID NO 384) 210-6345 sense CAAGCTGAAGCTGTTGGGTGTGGAC +963 to 987 (SEQ ID NO 385) 210-6346 antisense GGCGGTGCGGATGTAGAACATC+2211 to 2232 (SEQ ID NO 386) 210-5909 sense GGCTGGAACCTGTACGTTTG+2101 to 2120 (SEQ ID NO 387)  210-5910 antisense TCGTCGATGCTGCAGATATT+2556 to 2575 (SEQ ID NO 388) KPORF-38 210-5911 senseCCGCTTCGTCACTGTTGAG   −44 to −26 (SEQ ID NO 389)  210-5912 antisenseTGACTGACAAAGGCGGAGAG  +652 to +671 (SEQ ID NO 390) 210-5913 senseCGCTGAACGTCGATAATGTC  +599 to +618 (SEQ ID NO 391) 210-5914 antisenseGAACCACCTCGAGTTTCACC +1296 to +1315 (SEQ ID NO 392) 210-5915 senseCGCGCGATCTCTATCGTC +1211 to +1228 (SEQ ID NO 393) 210-5916 antisenseGAGACCGGAGATCGCTTTTT +2009 to +2028 (SEQ ID NO 394 210-5917 senseGCCGCGTTTGATATCGTCTA +1969 to +1988 (SEQ ID NO 395) 210-5918 antisenseCGTTTTAACTCGTCGCCATC +2881 to +2900 (SEQ ID NO 396) 210-5919 senseGTGGATAGCGGGGTACTGAA +2728 to +2747 (SEQ ID NO 397) 210-2920 antisenseGCCCCTCTCTATCCCATAGC +3756 to +3775 (SEQ ID NO 398) 210-5921 senseTAAAGGCGCTGGGCATTAG +3650 to +3668 (SEQ ID NO 399) 210-5922 antisenseGGCTGACTGCCGGTATTACT +4459 to +4478 (SEQ ID NO 400) 210-5923 senseAACCAACGCCTTGTTCCTT +4311 to +4329 (SEQ ID NO 401) 210-5924 antisenseGTGACCGGATAACGCCAGAC +5176 to +5195 (SEQ ID NO 402) KPORF-39 210-5925sense GTAAGGACATGCAGGAGATG  −132 to −113 (SEQ ID NO 403) 210-5926antisense GTAATATGGCGACGGTCTT  +892 to +910 (SEQ ID NO 404) KPORF-60210-5927 sense CATTAGGCTAGTCGTTCTCG   −54 to −35 (SEQ ID NO 405)210-5928 antisense TACGTTCCTGTTACCGTGTC  +851 to +870 (SEQ ID NO 406)KPORF-65 210-6564 sense GACGTCAGTTTACTGGTAGGC  −206 to −186(SEQ ID NO 407) 210-6565 antisense TCTTTCAGCTGACGAATGAC  +703 to +722(SEQ ID NO 408) 210-6566 sense CTCGTAATAACGCTCTATACCC  +624 to +645(SEQ ID NO 409) 210-6567 antisense CAATAGCCGGAATGGATA +1518 to +1535(SEQ ID NO 410) 210-6568 sense CTGATGATCCTGATTAACGAC +1405 to +1425(SEQ ID NO 411) 210-6569 antisense  CGATTTTACGCTCCATCAT +2034 to +2052(SEQ ID NO 412)

TABLE 8 Gene conservation of KPORF-13. Amino Strains Strains StrainsStrains Refer- Align- acid in with with with with ence ment Refer- AA AAAA AA respective respective respective respective position position encechange¹ change² change³ change⁴ change¹ change² change³ change⁴ 2 2 K NR S T 313, i202/94, 6613, 1680/49 1470 i221/94 5758, 4463/52 3 3 M L VD5050 1470 4 4 K I N D5050 1470, i221/94 5 5 L F 1470, i221/94 6 6 T P1470, i221/94 7 7 A T i202/94 10 10 S I 1470, i221/94 11 11 G V 1470,1680/49, i221/94 12 12 M I A5054, 919, Mich61, 2069/49, i224/94,5710/52, 708, 370, 5053 13 13 I M i202/94, 4463/52 14 14 L P i221/94 1515 S A i202/94, 6613, 5758, 4463/52 17 17 S L 919 18 18 A P 919, 5281 2424 T V 6613, 5758 27 27 D E 6613, 5758 29 29 M L 4463/52 30 30 V D 6613,4463/52 56 56 A T Aerogenes4140, 1470, 1996/49, 5711/52 84 84 D N1754/49 88 88 R H 1680/49, 5281 95 95 K Q 919, 1754/49, i202/94,1680/49, 5281, i224/94, 4463/52 107 107 D A D5050, E5051, Aerogenes4140,1470, Mich61, 1754/49, i202/94, 1996/49, 6613, 5758, 7749, i224/94,4463/52, 5711/52, 5053 122 122 N D i202/94, 4463/52 125 125 N Y 2005/49,Friedländer 204 183 183 A T 2005/49, Friedländer 204 249 249 K Qi202/94, 4463/52 260 260 K Q 6613, 5758 261 261 P S 4463/52 330 330 V IL NCTC8172 i202/94, 6613,5758, 4463/52 331 331 L V 1680/49 333 333 N H6613, 5758 337 337 V A 1680/49 338 338 D I 1680/49 340 340 L V 1680/49341 341 F V 1680/49 344 344 F L 6613 346 346 D A 1470, 1680/49, 6613 351351 F L 1680/49 352 352 L G R 1680/49, i221/94 6613 353 353 D H N 52811680/49 355 355 K I N 1680/49 5281 356 356 R H L Q A5054, D5050, 1470,i225/94, E5051, 919, 1680/49, Aerogenes4140, 1754/49, 6168, i221/942005/49, 1702/49, 7749 1996/49, 2002/49, 6613, 5758, 7824, 6258,3985/51, 5711/52, 708, 370, Friedländer 204 ^(1,2,3,4)observed aminoacid at respective position in any of the sequenced genes of therespective Klebsiella sp. strains in reference to Klebsiella pneumoniaeMGH78578.

TABLE 9 Gene conservation of KPORF-21. Amino Strains Strains StrainsStrains Refer- Align- acid in with with with with ence ment Refer- AA AAAA AA respective respective respective respective position position encechange¹ change² change³ change⁴ change¹ change² change³ change⁴ 1 1 V L5725y, 264-1 2 2 P T 264-1 5 5 E D B5055, 2005/49, 1702/49, 7824,i224/94 6 6 P A S T 7824 265-1, 5725y, 264-1 889 7 7 S T 889 12 12 G D5725y, 264-1 36 36 R G i262/94 39 39 V A D i262/94, i202/94, 5725y,Stanley, 4463/52, 5710/52 264-1, 265-1, 889 43 43 M I 2005/49 44 44 S G265-1, 889 52 52 D N 5725y 53 53 V A 919, 313, 6613, 5758, 6258, 6168,3985/51, i221/94, 5845/52 54 54 T I 4463/52 56 56 D E i262/94, i202/94,5725y, 1756/51, Stanley, 4463/52, 264-1, 265-1, 889 58 58 L V 5725y,264-1, 265-1, 889 59 59 E D 5725y, 264-1, 265-1, 889 60 60 Q K N R 264-15725y 265-1, 889 63 63 A E 5725y, 264-1 68 68 N K 2005/49 74 74 N E H Q5725y, 264-1 1756/51, 265-1, 889 Stanley 78 78 G R 5725y, 264-1, 265-1,889 85 85 S G i262/94 94 94 I V 5725y, 264-1 99 99 S K N 5725y, 264-1265-1, 889 104 104 A S 5725y, 264-1 106 106 Q E 5725y, 264-1, 265-1, 889107 107 V A D E T i202/94, 2482, 4463/52 1756/51, 265-1, 889 5725y,Stanley 264-1 108 108 V G I T 265-1, 889 5725y 264-1 109 109 D Ei202/94, 5725y, 2482, Stanley, 4463/52, 264-1, 265-1, 889 110 110 Q A R5725y, 264-1 265-1, 889 114 114 E K A5054, Mich61, 6613, 5758, 5845/52,708, 370, 5053 116 116 D E 5725y, 264-1, 265-1, 889 118 118 D Y 5710/52120 120 V A 5725y, 264-1, 265-1, 889 121 121 S L Q R 265-1, 889 5725y264-1 122 122 L F B5055 124 124 R K 5725y, 264-1, 265-1, 889 125 125 E Ai202/94, 4463/52 133 133 E D 265-1, 889 137 137 S T 5725y, 265-1, 889138 138 A V 5725y, 264-1, 265-1, 889 140 140 T A i202/94, 5725y,4463/52, 264-1, 265-1, 889 145 145 V I i202/94, 5725y, 4463/52, 264-1,265-1, 889 ^(1,2,3,4)observed amino acid at respective position in anyof the sequenced genes of the respective Klebsiella sp. strains inreference to Klebsiella pneumoniae MGH78578.

TABLE 10 Gene conservation of KPORF-32. Amino Strains Strains StrainsRefer- Align- acid in with with with ence ment Refer- AA AA AArespective respective respective position position ence change¹ change²change³ change¹ change² change³ 2 2 E V A5054, 6613 4 4 F I 6613 7 7 I VA5054, B5055, 6613, 5281 9 9 V I 4463/52 11 11 A D i243/94 12 12 G Ri243/94 19 19 A T i243/94 20 20 A P i243/94 21 21 Q D H i243/94 i221/94,5053 24 24 Q P i243/94 28 28 R H 5845/52 32 32 L V i243/94 34 34 N Ki243/94 36 36 K I R i243/94 6613 38 38 P M i243/94 45 45 S A i243/94 6060 T A A5054, B5055, i225/94, D5050, E5051, Aerogenes4140, 1015, Mich61,2069/49, i243/94, 2005/49, 1754/49, 1702/49, i202/94, 1996/49, 1680/49,2002/49, 6613, 5725y, 7824, 6258, 6168, i256/94, 7749, 2482, 5281,i224/94, 1756/51, Stanley, 3985/51, i221/94, 4463/52, 5710/52, 5711/52,5845/52, NCTC8172, 708, 370, 5053 75 75 A G 5725y 79 79 Q H 5725y 92 92A P 5725y 93 93 W R 5725y 99 99 G R 5725y 112 112 N T i202/94, 4463/52119 119 E G A5054 122 122 G D D5050, E5051, i243/94, 1756/51, Stanley124 124 Q E 2482 133 133 S L 5725y 136 136 R H S i224/94, 3985/51,NCTC8172 2069/49 151 151 M A T V i202/94, 4463/52 i225/94, 2005/49A5054, Aerogenes4140, 1015, Mich61, 1754/49, 1702/49, 1996/49, 1680/49,2002/49, 6613, 7824, 6258, 6168, i256/94, 7749, 2482, i221/94, 5710/52,5845/52, 708, 370, 5053 152 152 T A i225/94, 2005/49 176 176 V Ii202/94, 4463/52 211 211 S T 5725y 229 229 L M 5725y 238 238 P T 5725y240 240 V G 5725y 259 259 P T 5725y 262 262 D N i243/94, i202/94,1756/51, Stanley, 4463/52 264 264 D E i243/94, i202/94, 1756/51,Stanley, 4463/52 265 265 D E i202/94, 4463/52 268 268 N Y 5725y 272 272S G i202/94, 4463/52 276 276 N H 5725y 281 281 N K 5725y 286 286 Q P5725y 294 294 C R 5725y 312 312 R P 5725y 315 315 Q L 1754/49 316 316 TA i202/94, 4463/52 320 320 T K 5725y 354 354 M T i243/94 359 359 A G5725y 360 360 P S i243/94, 1756/51, Stanley 363 363 Y F S 4463/52 5725y367 367 E D 6613 368 368 V L M 2069/49 1015, i243/94, 6613, 5053 371 371V L 5725y 374 374 W R i243/94 377 377 G R i243/94 379 379 N K i243/94380 380 F T i243/94 382 382 W S i243/94 383 383 A P 6613 387 387 A Li243/94 388 388 W H i243/94 390 390 C S i243/94 391 391 A V Y D5050i243/94 394 394 L H i243/94 395 395 V M Q A5054 i243/94 397 397 G EAerogenes4140, 7824 ^(1,2,3)observed amino acid at respective positionin any of the sequenced genes of the respective Klebsiella sp. strainsin reference to Klebsiella pneumoniae MGH78578.

TABLE 11 Gene conservation of KPORF-37. Amino Strains Strains Refer-Align- acid in with with ence ment Refer- AA AA respective respectiveposition position ence change¹ change² change¹ change² 213 213 I V2005/49 218 218 T I i202/94, 4463/52 225 225 H R i202/94, 4463/52 254254 G E D5050, E5051 299 299 M T 1193 364 364 D H T 1470 NCTC8172 509509 D G 1680/49 513 513 E A i202/94, 4463/52 523 523 V I i243/94,1756/51 551 551 S T i243/94, i202/94, 1756/51, 4463/52 615 615 E KD5050, E5051 618 618 R Q 1680/49 727 727 E D A5054, B5055, D5050, E5051,1015, 919, 313, 1470, Mich61, 2069/49, 2005/49, 1754/49, 1996/49,1680/49, 2002/49, 6613, 5758, 6258, 6168, i256/94, i224/94, i221/94,5710/52, 5711/52, 370, Friedländer 204, 5053 731 731 K T i202/94,4463/52 792 792 C W 1680/49 803 803 A T A5054 827 827 D N 7749 845 845 EK Aerogenes4140 846 846 K N i243/94, 1756/51 847 847 A V i243/94,1756/51 ^(1,2)observed amino acid at respective position in any of thesequenced genes of the respective Klebsiella sp. strains in reference toKlebsiella pneumoniae MGH78578.

TABLE 12 Sequence length (in amino acids) as obtained by sequencing ofKlebsiella gene KPORF-38 from selected Klebsiella species and strains.All sequences were determined from the first codon (Start) to theindicated position. Sequence SEQ ID Strain name obtained (aa) NOMGH78578 1663 580 A5054 1254 581 i252/94 951 582 B5055 1403 583 i225/941454 584 D5050 1492 585 E5051 1463 586 Aerogenes4140 1492 587 1015 1457588 i262/94 1489 589 919 1476 590 313 1490 591 1470 1490 592 1193 1492593 Mich61 1490 594 2069/49 1491 595 2005/49 1489 596 1702/49 1419 5971996/49 1489 598 1680/49 1484 599 2002/49 1489 600 6613 1482 601 57581240 602 7824 1492 603 6258 1423 604 6168 1491 605 i256/94 1489 606 77491492 607 2482 1492 608 5281 1492 609 i224/94 1492 610 i221/94 1487 6115710/52 1489 612 5711/52 1490 613 NCTC8172 1252 614 708 949 615Friedländer 204 1244 616 5053 1474 617

TABLE 13 Gene conservation of KPORF-39. Amino Strains Strains Refer-Align- acid in with with ence ment Refer- AA AA respective respectiveposition position ence change¹ change² change¹ change² 1 1 V M 264-1 2 2D E 264-1, 265-1, 889 45 45 N S 265-1, 889 50 50 L F 265-1 52 52 P Si256/94 58 58 V L 264-1, 265-1, 889 82 82 S N 6258, 3985/51 96 96 I L264-1, 265-1, 889 117 117 I L 265-1, 889 119 119 A S 264-1 123 123 A Ti252/94, i262/94, i243/94, 1756/51, Stanley, 4463/52 126 126 S T 265-1,889 157 157 P A 264-1, 265-1, 889 159 159 D S 265-1, 889 161 161 T A264-1 176 176 L V 264-1 182 182 T S 265-1, 889 193 193 S N 265-1, 889199 199 V S 264-1, 265-1, 889 201 201 T A 264-1, 265-1, 889 213 213 L M6613, 5758, 5710/52 232 232 M I 265-1, 889 234 234 A S i262/94, i243/94,1756/51, Stanley 236 236 K A 264-1, 265-1, 889 237 237 A P T 1756/511470 241 241 N K 264-1, 265-1, 889 243 243 N K i202/94, 4463/52 245 245D E 265-1, 889 245 246 — Q 889 ^(1,2)observed amino acid at respectiveposition in any of the sequenced genes of the respective Klebsiella sp.strains in reference to Klebsiella pneumoniae MGH78578.

TABLE 14 Gene conservation of KPORF-60. Amino Strains Strains StrainsRefer- Align- acid in with with with ence ment Refer- AA AA AArespective respective respective position position ence change¹ change²change³ change¹ change² change³ 5 5 F L 265-1, 889 21 21 H Q 265-1, 88924 24 N D E T D5050, 264-1 265-1, 889 Stanley 26 26 T Q 265-1, 889 28 28V I 265-1, 889 66 66 T A I 265-1, 889 6613 95 95 D E 265-1, 889 117 117N K 265-1, 889 138 138 S G D5050, 264-1, 265-1, 889 192 192 N H 265-1,889 194 194 K Q 265-1, 889 200 200 E D D5050, 264-1, 265-1, 889 206 206N S 265-1, 889 i252/94, D5050, i262/94, i243/94, i202/94, 1996/49,1756/51, 231 231 D E Stanley, 4463/52, 264-1, 265-1, 889^(1,2,3)observed amino acid at respective position in any of thesequenced genes of the respective Klebsiella sp. strains in reference toKlebsiella pneumoniae MGH78578.

TABLE 15 Gene conservation of KPORF-65. Amino Reference Alignment acidin AA Strains with position position Reference change¹ respectivechange¹ 36 36 I V i252/94, i262/94, i243/94, 1756/51, Stanley 48 48 D Ni252/94, i262/94, i243/94, 1756/51, Stanley 61 61 L P E5051 100 100 Q Hi202/94 115 115 D E i202/94 125 125 R P i202/94 128 128 R A i202/94 144144 A V i202/94 186 186 D H i202/94 215 215 E D i202/94 283 283 I Vi252/94, i262/94, i243/94, 1756/51, Stanley 303 303 S R Stanley 308 308A G i252/94, i262/94, i243/94, i202/94, 1756/51, Stanley, 4463/52 364364 E D i252/94, i262/94, i243/94, 1756/51, Stanley 376 376 G D E5051389 389 D G Friedländer 204 405 405 S A i202/94, 4463/52 422 422 S Ai202/94, 4463/52 471 471 I L A5054 481 481 P S 5725y 497 497 V INCTC8172 511 511 Y F 5725y 516 516 K Q Aerogenes4140 520 520 Y Fi252/94, D5050, i262/94, i243/94, i202/94, 5725y, 1756/51, Stanley,4463/52 565 565 S A D5050, 5725y 589 589 M I D5050, 5725y 619 619 T ND5050, 5725y ¹observed amino acid at respective position in any of thesequenced genes of the respective Klebsiella sp. strains in reference toKlebsiella pneumoniae MGH78578.

TABLE 16 Overview over the antigen fragments used for protectionexperiments (Example 8) Seq-ID of Seq-ID of full Location in fullfragment length length Fragment name DNA/protein DNA/protein aa from-toKPORF-02.1 1/188 18/205  2-130 KPORF-13.1 2/189 29/216 26-356 KPORF-20.13/190 36/223  2-180 KPORF-21.1 4/191 37/224  1-168 KPORF-32.1 375/376 48/235 23-397 KPORF-37.1 5/192 53/240  2-420 KPORF-37.2 6/193 53/240414-847  KPORF-38.2 7/194 54/241 582-1099 KPORF-39.1 8/195 55/242  1-245KPORF-44.1 9/196 60/247 24-703 KPORF-49.1 10/197  65/252 23-328KPORF-60.1 11/198  76/263 23-248 KPORF-64.1 12/199  80/267  2-335KPORF-65.1 13/200  81/268 38-633 KPORF-66.1 14/201  82/269 26-742KPORF-78.1 15/202  94/281 26-429 KPORF-82.1 16/203  98/285  1-632

EXAMPLES Example 1 General Screening Procedure for the Identification ofthe Peptides According to the Present Invention

The approach, which has been employed for the present invention, isbased on the interaction of proteins or peptides encoded by K.pneumoniae with the antibodies present in human sera. The antibodiesproduced against K. pneumoniae by the human immune system and present inhuman sera are indicative of the in vivo expression of the antigenicproteins and their immunogenicity. In addition, the antigenic proteinsas identified by the bacterial surface display expression librariesusing pools of pre-selected sera, are processed in a second and thirdround of screening by individual selected or generated sera. Thus thepresent invention supplies an efficient, relevant, comprehensive set ofantigens as a pharmaceutical composition, especially a vaccinepreventing infections caused by K. pneumoniae .

In the antigen identification program for identifying a comprehensiveset of antigens according to the present invention, at least twodifferent bacterial surface expression libraries from K. pneumoniae arescreened with several serum pools or plasma fractions (antibody pools).The antibody pools are derived from a serum collection, which has beentested against antigenic compounds of K. pneumoniae, such as whole cell,total extracts. Preferably, four pools of sera (with 17 individualsamples) are used. Sera determined to have high ELISA titre have toreact with multiple proteins in immunoblotting in order to be consideredhyperimmune and therefore relevant in the screening method applied forthe present invention.

The expression libraries as used in the present invention should allowexpression of all potential antigens, e.g. derived from all secreted andsurface proteins of K. pneumoniae. Bacterial surface display librarieswill be represented by a recombinant library of a bacterial hostdisplaying a (total) set of expressed peptide sequences of K. pneumoniaeon two selected outer membrane proteins (LamB and FhuA) at the bacterialhost membrane (Georgiou, G., 1997); (Etz, H. et al., 2001). One of theadvantages of using recombinant expression libraries is that theidentified antigens may be instantly produced by expression of thecoding sequences of the screened and selected clones expressing theantigens without further recombinant DNA technology or cloning stepsnecessary.

The comprehensive set of antigens identified by the described programaccording to the present invention is analyzed further by one or moreadditional rounds of screening. Therefore individual antibodypreparations or antibodies generated against selected peptides, whichwere identified as immunogenic are used. According to a preferredembodiment the individual antibody preparations for the second round ofscreening are derived from healthy adults and/or challenged adults whoshow an antibody titre above a certain minimum level, for example anantibody titre being higher than 80 percentile, preferably higher than90 percentile, especially higher than 95 percentile of the human(patient or healthy individual) sera tested. Using such high titreindividual antibody preparations in the second screening round allows avery selective identification of the antigens and fragments thereof fromK. pneumoniae .

Following the comprehensive screening procedure, the selected antigenicproteins, expressed as recombinant proteins or in vitro translatedproducts, in case it can not be expressed in prokaryotic expressionsystems, or the identified antigenic peptides (produced synthetically)are tested in a second screening by a series of ELISA and Westernblotting assays for the assessment of their immunogenicity with a largehuman serum collection (minimum ˜20 healthy and patients sera).

It is important that the individual antibody preparations (which mayalso be the selected serum) allow a selective identification of the mostpromising candidates of all the antigens from all the promisingcandidates from the first round. Therefore, preferably at least 10individual antibody preparations (i.e. antibody preparations (e.g. sera)from at least 10 different individuals exposed to the chosen pathogen)should be used in identifying these antigens in the second screeninground. Of course, it is possible to use also less than 10 individualpreparations, however, selectivity of the step may not be optimal with alow number of individual antibody preparations. On the other hand, if agiven antigen (or an antigenic fragment thereof) is recognized by atleast 10 individual antibody preparations, preferably at least 30,especially at least 50 individual antibody preparations, identificationof the antigen is also selective enough for a proper identification.Serum-reactivity may of course be tested with as many individualpreparations as possible (e.g. with more than 100 or even with more than1,000).

Therefore, the relevant portion of the serum-reactive antibodypreparations according to the method of the present invention shouldpreferably be at least 10, more preferably at least 30, especially atleast 50 individual antibody preparations. Alternatively (or incombination) antigens may preferably be also identified with at least20%, preferably at least 30%, especially at least 40% of all individualantibody preparations used in the second screening round.

According to a preferred embodiment of the present invention, the serafrom which the individual antibody preparations for the second round ofscreening are prepared (or which are used as antibody preparations), areselected by their titre against K. pneumoniae (e.g. against apreparation of this pathogen, such as a lysate, cell wall components andrecombinant proteins). Preferably, some are selected with an IgG titreabove 1,000 U, especially above 5,000 U (U=units, calculated from theOD_(405nm) reading at a given dilution) when the whole organism (totallysate or whole cells) is used as antigen in the ELISA.

The antibodies produced against K. pneumoniae by the human immune systemand present in human sera are indicative of the in vivo expression ofthe antigenic proteins and their immunogenicity. The recognition oflinear epitopes recognized by serum antibodies can be based on sequencesas short as 4-5 amino acids. Of course it does not necessarily mean thatthese short peptides are capable of inducing the given antibody in vivo.For that reason the defined epitopes, polypeptides and proteins arefurther to be tested in animals (mainly in mice) for their capacity toinduce antibodies against the selected proteins in vivo.

The preferred antigens are located on the cell surface or secreted, andare therefore accessible extracellularly. Antibodies against cell wallproteins are expected to serve multiple purposes: to inhibit adhesion,to interfere with nutrient acquisition, to inhibit immune evasion and topromote phagocytosis (Hornef, M. et al., 2002). Antibodies againstsecreted proteins are beneficial in neutralisation of their function astoxin or virulence component. It is also known that bacteria communicatewith each other through secreted proteins. Neutralizing antibodiesagainst these proteins will interrupt growth-promoting cross-talkbetween or within infection causing pathogen species. Bioinformaticanalyses (signal sequences, cell wall localisation signals,transmembrane domains) proved to be very useful in assessing cellsurface localisation or secretion. The experimental approach includesthe isolation of antibodies with the corresponding epitopes and proteinsfrom human serum, and the generation of immune sera in mice against(poly) peptides selected by the bacterial surface display screens. Thesesera are then used in a third round of screening as reagents in at leastone of the following assays: cell surface staining of K. pneumoniaegrown under different conditions (FACS or microscopy), determination ofneutralizing capacity (toxin, adherence), and promotion of opsonizationand phagocytosis (in vitro phagocytosis assay).

For that purpose, bacterial E. coli clones are directly injected intomice and immune sera are taken and tested in the relevant in vitroassay. Alternatively, specific antibodies may be purified from human ormouse sera using peptides or proteins as substrate.

According to the antigen identification method used herein, the presentinvention can surprisingly provide a set of comprehensive novel nucleicacids and novel antigens, variants and fragments thereof of K.pneumoniae, among other things, as described herein. The nucleotidesequences according to the present invention encoding antigenspreferably have a nucleotide sequence which is individually set forth inSeq ID Nos 1 to 187 and Seq ID No 375, whereby the corresponding encodedamino acid sequences preferably have an amino acid sequence as set forthin Seq ID Nos 188 to 374 and Seq ID No 376.

All linear fragments of a particular antigen may be identified byanalysing the entire sequence of the protein antigen by a set ofpeptides overlapping by 1 amino acid with a length of at least 10 aminoacids. Subsequently, non-linear epitopes can be identified by analysisof the protein antigen with hyperimmune sera using the expressedfull-length protein or domain polypeptides thereof. Assuming that adistinct domain of a protein is sufficient to form the 3D structureindependent from the native protein, the analysis of the respectiverecombinant or synthetically produced domain polypeptide withhyperimmune serum would allow the identification of conformationalepitopes within the individual domains of multi-domain proteins. Forthose antigens where a domain possesses linear as well as conformationalepitopes, competition experiments with peptides corresponding to thelinear epitopes may be used to confirm the presence of conformationalepitopes.

Example 2 Characterization and Selection of Human Serum Samples Based onAnti-Klebsiella Antibodies and Preparation of Antibody ScreeningReagents Experimental Procedures

Enzyme linked immunosorbent assay (ELISA). ELISA plates (Maxisorb,Millipore) were coated with 5-10 μg/ml total protein diluted in coatingbuffer (0.1 M sodium carbonate pH 9.2). Two dilutions of sera (1,000×and 5,000×) were made in PBS-BSA. Highly specific Horse RadishPeroxidase (HRP)-conjugated anti-human IgG secondary antibodies(Southern Biotech) were used according to the manufacturer'srecommendations (dilution: 1,000×). Antigen-antibody complexes werequantified by measuring the conversion of the substrate (ABTS) tocoloured product based on OD_(405nm) readings by automatic ELISA reader(TECAN SUNRISE).

Preparation of total bacterial extracts. The K. pneumoniae strains Mich61 (K-type 15; O-type 4) and 708 (K-type 80; O-type 12) and theircapsule negative variants were grown overnight in Nutrient Broth (Difco234000) at 37° C. Cells were lysed by repeated freeze-thaw cycles,following incubation on dry ice/ethanol-mixture until frozen for 1 minand thawing at 37° C. for 5 min. The lyses procedure was repeated 3times, followed by sonication. After centrifugation at 4,000 rpm for 15min at 4° C., the supernatant contains whole cell extracts and wascollected. Pellets were discarded and protein concentration was measuredwith the Bradford assay using protein assay dye reagent concentrate(Bio-Rad Laboratories, Austria).

Purification of antibodies for genomic screening. Four to five sera perantibody pool were selected mainly based on their stronger reaction inELISA against the capsule negative strain against the ones with capsule.Antibodies against E. coli DH5alpha proteins were removed by incubatingthe heat-inactivated sera with whole cell E. coli DH5alpha cells(transformed with pHIE11, grown under the same condition as used forbacterial surface display). Highly enriched preparations of IgGs fromthe pooled, depleted sera were generated by protein G affinitychromatography, according to the manufacturer's instructions (UltraLinkImmobilized Protein G, Pierce). The efficiency of depletion andpurification was checked by ELISA measurements.

Results

The antibodies produced against K. pneumoniae by the human immune systemand present in human sera are indicative of the in vivo expression ofthe antigenic proteins and of their immunogenicity. These molecules areessential for the identification of individual antigens in the approachas described in the present invention, which is based on the interactionof the specific anti-bacterial antibodies and the corresponding K.pneumoniae peptides or proteins. To gain access to relevant antibodyrepertoires, human sera were collected from patients with septicaemiaand healthy exposed people.

50 acute phase serum samples and 100 convalescent serum samples werecollected from 100 donors and characterized for anti-K. pneumoniaeantibodies together with 49 sera taken from healthy individuals by aseries of immune assays. Primary characterization was done by ELISAusing total bacterial lysates for K. pneumoniae strains Mich 61 and 708and their respective capsule negative mutants. Antibody titers weremeasured and ELISA units calculated from serum dilutions in the linearrange of response. Sera were ranked based on the differential reactionin ELISA against lysates prepared from strains with and without capsule,which was used for the selection of sera to be included inantibody-pools. The reactivity of the sera used for the generation ofpools against bacterial lysates from K. pneumoniae is shown in FIGS. 1Aand B.

Selected sera were included in 4 different IgG pools (4-5 sera in eachpool) for antigen identification by bacterial surface display. IgGantibodies were purified from pooled sera by affinity chromatography anddepleted of E. coli DH5alpha-reactive antibodies to avoid background inthe bacterial surface display screens. The serum pools representinghealthy individuals are ICKp18 (IC38, IC40, IC76 and IC86) and ICKp19(IC88, IC89, IC92 and IC93) and from patients with septicaemia are PKp34(P3536.2, P3548, P3560, P3582 and P3583) and PKp35 (P3495.2, P3533.2,P3567 and P3576).

Example 3 Generation of Highly Random, Frame-Selected, Small-Fragment,Genomic DNA Libraries of K. pneumoniae Experimental Procedures

Preparation of genomic DNA from K. pneumoniae strain MGH78578 (ATCC700721). Cells from a 400 ml bacterial culture were harvested (5,000rpm, 20 min, room temperature), washed with 80 ml 50 mM Tris pH 7.4 andre-suspended in 10 ml 50 mM Tris pH 7.4/25% Sucrose/50 mM EDTA. Thesuspension was transferred to a fresh glass tube and Lysozyme (finalconcentration: 1.5 mg/ml) and SDS (final conc.: 2%) were added. The tubewas incubated on ice for cell lysis. Proteinase K (final concentration:0.1 mg/ml) was added and incubated for 10 min at 37° C., followed byPhenol/Chloroform (1:1) extraction, which was performed several times. Afinal extraction step was performed with Chloroform/Isoamylalcohol(1:24) to remove Phenol traces. The sample was treated with RNase A(final concentration: 10 μg/ml) for 1 h at room temperature andPhenol/Chloroform and Chloroform/Isoamylalcohol extractions wereperformed as described above. DNA in the remaining supernatant wasprecipitated by addition of 1/10th of the starting volume of 3 M NaAc(pH 5.3) and 2.5× of the volume of 99.5% Ethanol. After 1 h incubationat −20° C., the mixture was centrifuged (20,000 rpm, 15 min) and thepellet washed with 70% Ethanol. Finally, the pellet was dissolved inTE-buffer.

Preparation of Small Genomic DNA Fragments. Genomic DNA Fragments wereMechanically sheared into fragments ranging in size between 150 and 300by using a cup-horn sonicator (Bandelin Sonoplus UV 2200 sonicatorequipped with a BB5 cup horn, 10 sec. pulses at 100% power output) orinto fragments of size between 50 and 70 by mild DNase I treatment(Novagen). It was observed that sonication yielded a much tighterfragment size distribution when breaking the DNA into fragments of the150-300 by size range. However, despite extensive exposure of the DNA toultrasonic wave-induced hydromechanical shearing force, subsequentdecrease in fragment size could not be efficiently and reproduciblyachieved. Therefore, fragments of 50 to 70 by in size were obtained bymild DNase I treatment using Novagen's shotgun cleavage kit. A 1:20dilution of DNase I provided with the kit was prepared and the digestionwas performed in the presence of MnCl₂ in a 60 μl volume at 20° C. for 5min to ensure double-stranded cleavage by the enzyme. Reactions werestopped with 2 μl of 0.5 M EDTA and the fragmentation efficiency wasevaluated on a 2% TAE-agarose gel. This treatment resulted in totalfragmentation of genomic DNA into near 50-70 by fragments. Fragmentswere then blunt-ended twice using T4 DNA Polymerase in the presence of100 μM each of dNTPs to ensure efficient flushing of the ends. Fragmentswere used immediately in ligation reactions or frozen at −20° C. forsubsequent use.

Description of the vectors. The vector pMAL4.31 was constructed on apASK-IBA backbone (Skerra, A., 1994) with the beta-lactamase (bla) geneexchanged with the Kanamycin resistance gene. In addition, the bla genewas cloned into the multiple cloning site. The sequence encoding maturebeta-lactamase is preceded by the leader peptide sequence of ompA toallow efficient secretion across the cytoplasmic membrane. Furthermore asequence encoding the first 12 amino acids (spacer sequence) of maturebeta-lactamase follows the ompA leader peptide sequence to avoid fusionof sequences immediately after the leader peptidase cleavage site, sincee.g. clusters of positive charged amino acids in this region woulddecrease or abolish translocation across the cytoplasmic membrane(Kajava, A. et al., 2000). A SmaI restriction site serves for libraryinsertion. An upstream FseI site and a downstream NotI site, which wereused for recovery of the selected fragment, flank the SmaI site. Thethree restriction sites are inserted after the sequence encoding the 12amino acid spacer sequence in such a way that the bla gene istranscribed in the −1 reading frame resulting in a stop codon 15 byafter the NotI site. A +1 by insertion restores the bla ORF so thatbeta-lactamase protein is produced with a consequent gain of Ampicillinresistance.

The vector pMAL9.1 was constructed by cloning the lamB gene into themultiple cloning site of pEH1 (Hashemzadeh-Bonehi, L. et al., 1998).Subsequently, a sequence was inserted in lamB after amino acid 154,containing the restriction sites FseI, SmaI and NotI. The reading framefor this insertion was constructed in such a way that transfer offrame-selected DNA fragments excised by digestion with FseI and NotIfrom plasmid pMAL4.31 yields a continuous reading frame of lamB and therespective insert.

The vector pHIE11 was constructed by cloning the JhuA gene into themultiple cloning site of pEH1. Thereafter, a sequence was insertedinfhuA after amino acid 405, containing the restriction site FseI, XbaIand NotI. The reading frame for this insertion was chosen in a way thattransfer of frame-selected DNA fragments excised by digestion with FseIand NotI from plasmid pMAL4.31 yields a continuous reading frame of fhuAand the respective insert.

Cloning and evaluation of the library for frame selection. Genomic K.pneumoniae (strain MGH78578). DNA fragments were ligated into the SmaIsite of the vector pMAL4.31. Recombinant DNA was electroporated intoDH10B electrocompetent E. coli cells (GIBCO BRL) and transformantsplated on LB-agar supplemented with Kanamycin (50 μg/ml) and Ampicillin(50 μg/ml). Plates were incubated over night at 37° C. and coloniescollected for large scale DNA extraction. A representative plate wasstored and saved for collecting colonies for colony PCR analysis andlarge-scale sequencing. A simple colony PCR assay was used to initiallydetermine the rough fragment size distribution as well as insertionefficiency. From sequencing data the precise fragment size wasevaluated, junction intactness at the insertion site as well as theframe selection accuracy (3n+1 rule).

Cloning and evaluation of the library for bacterial surface display.Genomic DNA fragments were excised from the pMAL4.31 vector, containingthe K. pneumoniae library with the restriction enzymes FseI and NotI.The entire population of fragments was then transferred into plasmidspMAL9.1 (LamB) or pHIE11 (FhuA), which have been digested with FseI andNotI. Using these two restriction enzymes, which recognise an 8 by GCrich sequence, the reading frame that was selected in the pMAL4.31vector is maintained in each of the platform vectors. The plasmidlibrary was then transformed into E. coli DH5alpha cells byelectroporation. Cells were plated onto large LB-agar platessupplemented with 50 μg/ml Kanamycin and grown over night at 37° C. at adensity yielding clearly visible single colonies. Cells were thenscraped off the surface of these plates, washed with fresh LB medium andstored in aliquots for library screening at −80° C.

Results

Libraries for frame selection. Two libraries were generated for K.pneumoniae strain MGH78578 in the pMAL4.31 vector with sizes ofapproximately 70 and 300 bp, respectively. For each library, ligationand subsequent transformation of approximately 1 μg of pMAL4.31 plasmidDNA and 50 ng of fragmented genomic K. pneumoniae DNA yielded 3×10⁵ to2×10⁶ clones after frame selection. To assess the randomness of thelibraries, approximately 500 to 600 randomly chosen clones of eachlibrary were sequenced. The representative bioinformatic analysis of twolibraries (KPL50 and KPF300) showed that of clones corresponding tothese libraries only very few were present more than once. Furthermore,it was shown for the KPL50 library that the average insert size was 77bp, very close to the expected insert size (FIG. 2A). Regarding theKPF300 library, the average insert size was 187 by slightly shorter thanthe expected insert size (FIG. 2B).

Bacterial surface display libraries. The display of peptides on thesurface of E. coli required the transfer of the inserts from the KPL50and the KPF300 libraries from the frame selection vector pMAL4.31 to thedisplay plasmids pMAL9.1 (LamB) or pHIE11 (FhuA). Genomic DNA fragmentswere excised by FseI and NotI restriction and ligation of 5 ng insertswith 0.1 μg plasmid DNA and subsequent transformation into DH5alphacells resulted in 3×10⁵ to 2×10⁶ clones. The clones were scraped off theLB plates and frozen without further amplification.

Example 4 Identification of Highly Immunogenic Peptide Sequences from K.pneumoniae Using Bacterial Surface Displayed Genomic Libraries and HumanSerum Experimental Procedures

MACS screening. Approximately 2.5×10⁸ cells from a given library weregrown in 5 ml LB-medium supplemented with 50 μg/ml Kanamycin for 2 h at37° C. Expression was induced by the addition of 1 mM IPTG for 30 min.Cells were washed twice with fresh LB medium and approximately 2×10⁷cells re-suspended in 100 μl LB medium and transferred to an Eppendorftube.

10 to 20 μg of biotinylated, human IgGs purified from serum was added tothe cells and the suspension incubated overnight at 4° C. with gentleshaking 900 μl of LB medium was added, the suspension mixed andsubsequently centrifuged for 10 min at 6,000 rpm at 4° C. Cells werewashed once with 1 ml LB and then re-suspended in 100 μl LB medium. 10μl of MACS microbeads coupled to streptavidin (Miltenyi Biotech,Germany) were added and the incubation continued for 20 min at 4° C.Thereafter 900 μl of LB medium was added and the MACS microbead cellsuspension was loaded onto the equilibrated MS column (Miltenyi Biotech,Germany), which was fixed to the magnet. The MS columns wereequilibrated by washing once with 1 ml 70% EtOH and twice with 2 ml LBmedium.

The column was then washed three times with 3 ml LB medium. Afterremoval of the magnet, cells were eluted by washing with 2 ml LB medium.After washing the column with 3 ml LB medium, the 2 ml eluate was loadeda second time on the same column and the washing and elution processrepeated. The loading, washing and elution process was performed a thirdtime, resulting in a final eluate of 2 ml.

Cells selected after two rounds of selection were plated onto LB-agarplates supplemented with 50 μg/ml Kanamycin and grown over night at 37°C.

Evaluation of selected clones by sequencing and Western blot analysis.Randomly selected clones were grown overnight at 37° C. in 3 ml LBmedium supplemented with 50 μg/ml Kanamycin to prepare plasmid DNA usingstandard procedures. Sequencing was performed at MWG (Germany) or Agowa(Germany).

For Western blot analysis approximately 10 to 20 μg of total cellularprotein was separated by 10% SDS-PAGE and blotted onto HybondC membrane(Amersham Pharmacia Biotech, England). The LamB or FhuA fusion proteinswere detected using human serum as the primary antibody at a dilution ofapproximately 1:3,000 to 1:5,000 and anti-human IgG antibodies coupledto HRP at a dilution of 1:5,000 as secondary antibodies. Detection wasperformed using the ECL detection kit (Amersham Pharmacia Biotech,England). Alternatively, rabbit anti-FhuA or rabbit anti-LamB polyclonalimmune sera were used as primary antibodies in combination with therespective secondary antibodies coupled to HRP for the detection of thefusion proteins.

Results

Screening of bacterial surface display libraries by magnetic activatedcell sorting (MACS) using biotinylated Igs. The libraries KPL50 inpMAL9.1 and KPF300 in pHIE11 were screened with pools of biotinylated,human IgGs prepared from sera of uninfected healthy adults (IC38, IC40,IC76, IC86, IC88, IC89, IC92, and IC93) and patients with acute K.pneumoniae infections (septicaemia) (P3536.2, P3548, P3560, P3582,P3583, P3495.2, P3533.2, P3567 and P3576) (see Example 1: Preparation ofAntibodies from Human Serum). the Selection Procedure was Performed asdescribed under Experimental procedures. FIG. 3A shows a representativeexample of a screen with the KPL50 library and PKp34-IgGs. As can beseen from the colony count after the first selection cycle from MACSscreening, the total number of cells recovered at the end is drasticallyreduced from 2.2×10⁷ cells to approximately 1.2×10⁴ cells, and theselection without antibodies showed a more pronounced reduction in cellnumbers, showing that selection was dependent on K. pneumoniae specificantibodies (FIG. 3A). To evaluate the performance of the screen, 20selected clones were picked randomly and subjected to immunoblotanalysis with the screening PKp34-IgG pool (FIG. 3B). This analysisrevealed that a majority of selected clones showed reactivity withantibodies present in the relevant serum whereas the control strainexpressing FhuA without a K. pneumoniae specific insert did not reactwith the same serum. In general, the rate of reactivity was observed tolie within the range of 15 to 85%. Colony PCR analysis showed that allselected clones contained an insert in the expected size range (data notshown). Similar results were seen in screens with libraries from theother serum pools. As a second example, FIGS. 3C and D show the dataobtained with the large insert KPF300 and the PKp35-IgG antibody pool.One round of MACS selection resulted in the enrichment of cells only inthe presence, but not the absence of specific IgG (FIG. 3C), indicatingthat the selection was specific for the applied antibodies. The specificselection was then confirmed in the Western blot analysis of individualbacterial clones with the same PKp35-IgG antibody pool (FIG. 3D).

Subsequent sequencing of a larger number of randomly picked clones (600to 800) from each screen led to the identification of the gene and thecorresponding peptide or protein sequence that was specificallyrecognized by the human serum antibodies used for screening. Thefrequency with which a specific clone is selected reflects at least inpart the abundance and/or affinity of the specific antibodies in theserum used for selection and recognizing the epitope presented by thisclone. Table 1 summarizes the data obtained for all 8 performed screens.All clones that are presented in Table 1 have been verified byimmunoblot analysis using whole cellular extracts from single clones toshow the indicated reactivity with the pool of human serum used in therespective screen. As can be seen from Table 1, distinct regions of theidentified ORF are identified as immunogenic, since variably sizedfragments of the proteins are displayed on the surface by the platformproteins.

It is further worth noticing that many of the genes identified by thebacterial surface display screens encode proteins that are attached tothe surface of the bacterium. This is in accordance with the expectedrole of surface attached proteins in virulence of K. pneumoniae .

Example 5 Gene Distribution Studies with Highly Immunogenic ProteinsIdentified from K. pneumoniae Experimental Procedures

Gene distribution of antigens by PCR. An ideal vaccine antigen would bean antigen that is present in all, or the vast majority of strains ofthe target organism the vaccine is directed to. In order to establishwhether the genes encoding the identified K. pneumoniae antigens occurubiquitously in the relevant strains, PCR was performed on a series ofindependent bacterial isolates with primers specific for the gene ofinterest. Oligonucleotide sequences as primers were designed for allidentified ORFs yielding products of approximately 1,000 bp, if possiblecovering all identified immunogenic epitopes. Genomic DNA of all K.pneumoniae strains was prepared as described under Example 2. PCR wasperformed in a reaction volume of 25 μA using Taq polymerase (1 U), 200nM dNTPs, 10 pMol of each oligonucleotide and the kit according to themanufacturer's instructions (Invitrogen, The Netherlands). As standard,30 cycles (lx: 5 min. 95° C., 30×:30 sec. 95° C., 30 sec. 56° C., 30sec. 72° C., 1×4 min. 72° C.) were performed, unless conditions had tobe adapted for individual primer pairs.

Results

Identified genes encoding immunogenic proteins were tested by PCR fortheir presence in 46 different K. pneumoniae strains (Table 3). Alltogether 95 genes were analyzed. 40% (38/95) were detected in >90% ofstrains (>41/46), while 25% (24/95) were missing in >75% of the strains(<35/46) and therefore categorized as not sufficiently conserved. As anexample, FIG. 4 shows the PCR reaction for the K. pneumoniae KPORF-54antigen with all indicated 46 strains. As clearly visible, the gene ispresent in all strains analyzed. All results with the selected antigensare summarized in Table 3. Importantly, 80% of the identified antigenswere well conserved among the strains of Klebsiella pneumoniae analyzed,at least in the presence and size of the gene-specific PCR products,therefore selected for further studies to evaluate their vaccinecandidate potential.

Example 6 Validation of Peptides from K. pneumoniae by Peptide ELISA

Enzyme linked immunosorbent assay (ELISA). ELISA plates (Maxisorb,Millipore) were coated with 5-10 μg/ml total protein diluted in coatingbuffer (0.1 M sodium carbonate pH 9.2). Two dilutions of sera (400× and2,000×) were made in PBS-BSA. Highly specific Horse Radish Peroxidase(HRP)-conjugated anti-human IgG secondary antibodies (Southern Biotech)were used according to the manufacturer's recommendations (dilution:1,000×). Antigen-antibody complexes were quantified by measuring theconversion of the substrate (ABTS) to coloured product based onOD_(405nm) readings by automatic ELISA reader (TECAN SUNRISE). Themeasurements at 400× dilution were used for the calculation of theresults as displayed in Table 4.

Results

Immunogenicity in humans. The presence of specific antibodies in humansera was determined by peptide ELISA as summarized in Table 4. The humansera used for this analysis correspond to those that were included inthe various serum pools applied for the identification of antigens bythe bacterial surface display screens. Single or multiple peptides fromindividual antigens from K. pneumoniae MGH78578 were analyzed and manyof these were shown to be immunogenic in humans. It is evident that someof the selected peptides are highly reactive with many or all of theinvestigated human sera (e.g. ORF-26.01 or ORF-81.01), while othersshowed intermediate or low reactivities. For those antigens for whichthe selected epitope encompassed more than 30 amino acids, multiplepeptides were designed with an overlap of 5 to 6 amino acids. For someof the antigens, it was observed that these multiple peptides from thesame antigen showed different reactivities, further delineating theimmunogenic region of the respective antigen (e.g. KPORF-27 orKPORF-42). These experiments confirmed that many of the identifiedepitopes/proteins are highly immunogenic in humans, indicating that theyare expressed by the pathogen during infection and capable of inducing astrong immune response.

Example 7 Surface Binding to K. pneumoniae of Immune Sera Obtained fromMice Immunized with Highly Immunogenic Proteins/Peptides from K.pneumoniae Displayed on the Surface of E. coli

Experimental procedures. FACS analysis. The K. pneumoniae strains A5054and Friedländer 204 were inoculated from a glycerol stock into 5 ml THBmedium and incubated over night at 37° C. The overnight culture wasreinoculated by adding 200 μA into 10 ml fresh THB medium and incubateduntil an OD₆₀₀ of approximately 0.6 was reached (−5×10⁸ cells/ml). Thebacteria were pelleted by centrifugation at 4,000 rpm for 5 min andwashed twice with 2 ml HBSS. The final pellet was resuspended in HBSSwith 0.5% BSA to give a cell density of 5×10⁶ cells/ml. To 100 μAbacteria, 5 μA immune serum was added and incubated for 45 min on ice.Bacteria were pelleted by centrifugation at 1,000 g for 4 min and washedonce with 1 ml HBSS with 0.5% BSA and resuspended in 100 μA HBSS with0.5% BSA. To the opsonised bacteria conjugated anti-mouse antibody wereadded at a dilution according to the manufacturer's recommendations(diluted in 100 μl). Secondary staining was performed on ice for 45 minin darkness. At the end of the incubation, the samples are centrifugedat 5,000 rpm for 3 min, washed with 1 ml HBSS and resuspended in 1 ml ofHBSS-2% paraformaldehyde. Samples were vortexed, fixed overnight andanalyzed by flow cytometry.

Results

Surface binding to K. pneumoniae cells. The presence of antibodiesrecognising surface proteins on K. pneumoniae A5054 and Friedländer 204were tested in FACS analysis. Of 103 antigens represented by differentsera, 36 showed a significant shift in the FACS analysis compared to thebuffer control as summarized in Table 5. These in vitro experimentsindicate that in in vitro cultured K. pneumoniae cells these 36 antigenswere expressed on the surface. As an example, FIG. 5 shows the FACSstaining for the K. pneumoniae KPORF-28, KPORF-82 and KPORF-02 antigenswhich are representatives of the categories “+”, “++” and “+++”,respectively.

Example 8 Antigens Induce Protective Immune Responses Against LethalSepsis Induced by K. pneumoniae Experimental Procedures

Expression and purification of recombinant Klebsiella pneumoniaeproteins. Cloning of genes/DNA fragments: The gene/DNA fragment ofinterest was amplified from genomic DNA of K. pneumoniae MGH78578 by PCRusing gene specific primers. Apart from the gene specific part, theprimers had restriction sites that aided in a directional cloning of theamplified PCR product. The gene annealing (specific) part of the primerranged between 15-30 bases in length. The PCR products obtained weredigested with the appropriate restriction enzymes and cloned into thepET28b (+) vector (Novagen) for His-tagged proteins. Once therecombinant plasmid was confirmed to contain the gene of interest, E.coli BL21-CodonPlus® cells (Stratagene) that served as expression hostwere transformed. The inserts were sequenced. The nucleotide sequencesand the amino acid sequences of the gene specific fragments are listedunder the respective sequence identification numbers (Seq ID Nos DNA,protein): KPORF-02.1:1, 188; KPORF-13.1:2, 189; KPORF-20.1:3, 190;KPORF-21.1:4, 191; KPORF-32.1:375, 376; KPORF-37.1:5, 192; KPORF-37.2:6,193; KPORF-38.2:7, 194; KPORF-39.1:8, 195; KPORF-44.1:9, 196;KPORF-49.1:10, 197; KPORF-60.1:11, 198; KPORF-64.1:12, 199;KPORF-65.1:13, 200; KPORF-66.1:14, 201; KPORF-78.1:15, 202;KPORF-82.1:16, 203 (see also Table 16). Expression and purification ofproteins. E. coli BL21-CodonPlus® cells harboring the recombinantplasmid of choice were grown into log phase in the required culturevolume. Once an OD_(600nm) of 0.6 was reached the culture was inducedwith 0.5 mM IPTG for 3 hours at 37° C. The cells were harvested bycentrifugation, lysed by a combination of the freeze-thaw methodfollowed by disruption of cells with ‘Bug-buster®, (Novagen). The lysatewas separated by centrifugation into soluble (supernatant) and insoluble(pellet) fractions. Depending on the location of the protein differentpurification strategies were applied. A) If the His-tagged protein wasin the soluble fraction, protein purification was done by binding thesupernatant to Ni-Sepharose beads (Ni-Sepharose™ 6 Fast Flow, GEHealthcare). Due to the presence of the hexa Histidine (6×HIS) at theC-terminus of the expressed protein, it bound to the Ni-Sepharose whilethe other contaminating proteins were washed from the column by washbuffer. The protein was eluted by 500 mM Imidazole in 20 mM NaH₂PO₄, 0.5mM NaCl buffer at pH 7.4. The eluate was concentrated, assayed byBradford for protein concentration and checked by SDS-PAGE and Westernblot. B) If the protein was present in the insoluble fraction, thepellet was solubilized in suitable buffer containing 8 M Urea andapplied onto the Ni-NTA column under denaturing conditions (in buffercontaining 8 M Urea) using the same materials and procedure as mentionedabove. Contaminating proteins were washed from the column by wash bufferwithout urea. Refolding of the His-tagged protein was performed whilethe protein was immobilized on the Ni-NTA matrix. After renaturation,proteins were eluted by the addition of 500 mM Imidazole. The eluate wasdialyzed to remove traces of urea and concentrated if the volume waslarge, checked by SDS-PAGE and measured by the Bradford method.

Animal Protection Studies

Animals: CD-1 female mice (6-8 weeks) were used.

Active immunization (subcutaneous route): 50 μg of recombinant proteinsas listed in Table 16 adjuvanted with either Complete Freund's adjuvant(CFA), Alum, or IC31® were injected subcutaneously into CD-1 mice. Ondays 14 and 28, mice were boosted with the same amount of protein andadjuvant (except that Incomplete Freund's adjuvant (IFA) was used ratherthan CFA). Mice immunized with K. pneumoniae B5055 lysate served as apositive control, while mice immunized with PBS combined with adjuvantonly served as a negative control. Antibody titres were measured at day35 by ELISA using the respective recombinant proteins.

Passive immunization (intraperitoneal route): 150 μA of hyper-immunerabbit serum raised against individual K. pneumoniae recombinant proteinantigens as listed in Table 16 was injected intraperitoneally (IP) intoCD-1 mice, one to three hours prior to IP bacterial challenge. Antibodytitres of the sera used for immunization were measured using therespective recombinant proteins.

Bacterial challenge: Freshly grown K. pneumoniae strain B5055 was used.In order to determine the viable cell numbers present in the bacterialinoculum, CFU were determined by plating dilutions of the inoculum ontoblood agar plates. 10³ CFU were applied intraperitoneally. Protectionconferred by immunization was measured using a bacteraemia/sepsis modelin which survival rates were followed for 2 weeks post-challenge, andsurvival was expressed as a percentage of the total number of animals(10 mice/group).

Results Active Immunization Experiments

As one of the main target indications for a preventive vaccine in humansis sepsis, an intraperitoneal challenge model for the evaluation ofcandidate antigens was employed and pre-selected K. pneumoniae antigenswere tested for showing protection in this murine sepsis/lethalitymodel. Protection has so far been observed for two distinct proteins inthe intraperitoneal challenge model. As protection against K. pneumoniaechallenge is mediated by antibodies, immunizations were performed usingCFA/IFA as adjuvant in order to obtain the highest levels of antibodies.As can be seen in two independent experiments depicted on FIG. 6A andFIG. 6B/C, proteins KPORF-21.1 and KPORF-60.1 gave 100%, KPORF-38.2 andKPORF-39.1 90% and 60% protection, respectively. These antigens have notpreviously been shown to protect against K. pneumoniae infection.

Partial protection was observed for 9 ORFs; KPORF-02.1 (30%), KPORF-13.1(40%), KPORF-20.1 (40%), KPORF-37.1 (20%), KPORF-37.2 (20%), KPORF-44.1(30%), KPORF-64.1 (40%), KPORF-65.1 (50%), and KPORF-66.1 (40%) (FIGS.6A, B, and C).

Recombinant proteins KPORF-13.1 and KPORF-37.1 were re-tested in thesame model. In a single experiment, immunization with KPORF-13.1 gaveprotection of 70% and KPORF-37.1 gave protection of 50% against K.pneumoniae IP challenge (FIG. 6D).

In addition, two further recombinant proteins, KPORF-32.1 andKPORF-39.1-IB (protein is the same as KPORF-39.1 but was immunized asinclusion bodies), were tested in the same model and gave protectionlevels of 50% and 40% respectively (FIG. 6E). Immunization with thepositive control, K. pneumoniae B5055 lysate, consistently gave 100%protection in all experiments.

The eight K. pneumoniae recombinant proteins which demonstratedprotection in the previously described set of experiments whenadjuvanted with CFA/IFA, were re-tested in active immunizationexperiments using either Alum or IC31® as adjuvant. In three independentexperiments in which Alum was used as adjuvant, KPORF-13.1 andKPORF-21.1 gave 60% (FIG. 7A), 50%, and 40% protection respectively.KPORF-32.1 gave 80% (FIG. 7A), 75%, and 70% protection respectively. Inthree independent experiments, KPORF-39.1-IB gave 100% protection (FIG.7B, single experiment shown). KPORF-37.1 gave 40% protection (FIG. 7A),KPORF-38.2 gave 60% (FIG. 7B), and KPORF-65.1 gave 50% (FIG. 7B)protection consistently in three independent experiments (singleexperiments shown). KPORF-60.1 gave 67% (FIG. 7B), 58%, and 50%protection in three independent experiments.

In three independent experiments in which IC31® was used as adjuvant,protein KPORF-13.1 gave 40% (FIG. 8A), 35%, and 30% protectionrespectively. KPORF-21.1 gave 60% (FIG. 8B), 55%, and 50% protection.Immunization with KPORF-32.1 resulted in only 22% (FIG. 8B), 16%, and10% survival. KPORF-37.1 gave 30% (FIG. 8B, single experiment shown)protection in three independent experiments. KPORF-38.2 gave 60% (FIG.8A), 55%, and 50% protection in three independent experiments.KPORF-39.1-IB gave 50% (FIG. 8A), 40%, and 30% protection respectively.KPORF-60.1 gave 63% (FIG. 8A), 59%, and 56% protection respectively.Immunization with KPORF-65.1 resulted in 50% (FIG. 8B), 35%, and 20%survival respectively.

Passive Immunization Experiments

Hyper-immune rabbit sera were raised individually against K. pneumoniaeB5055 lysate, PBS, and the eight individual K. pneumoniae recombinantproteins demonstrating protection in active immunization experiments;KPORF-13.1, KPORF-21.1, KPORF-32.1, KPORF-37.1, KPORF-38.2, KPORF-39.1,KPORF-60.1, and KPORF-65.1 In single experiments, passive immunizationof mice with KPORF-60.1 sera gave 60% protection (FIG. 9A) andimmunization with KPORF-37.1 sera gave 40% protection against K.pneumoniae challenge (FIG. 9B). 100% protection was observed in all miceimmunized with K. pneumoniae B5055 lysate sera. No survival was observedin mice immunized with PBS sera (FIGS. 9A & B). 20% survival wasobserved in mice immunized with KPORF-32.1 sera (FIG. 9B). No protectionwas observed in mice immunized with sera raised against the other fiveproteins KPORF-13.1, KPORF-21.1, KPORF-38.2, KPORF-60.1 (FIG. 9A),KPORF-65.1 (FIG. 9B).

These antigens have not previously been shown to protect against K.pneumoniae infection.

Example 9 Gene conservation of Klebsiella Antigens

Preparation of Klebsiella sp. Genomic DNA

7 ml DIFCO0001 medium were inoculated with the respective strain ofKlebsiella sp. from a frozen stab and grown without shaking at 37° C.overnight. 2 ml of the culture were then harvested by centrifuging at13,000 rpm in a Biofuge fresco (Heraeus) for 5 min and the supernatantwas removed. DNA was isolated from the bacterial cell pellets followingthe protocol of Wizard® Genomic DNA Purification Kit (Promega). The DNApellets were finally dried on air and dissolved in 70 μl ddH₂O.

PCR amplification of Klebsiella sp. antigens PCR was performed on aseries of independent Klebsiella sp. isolates (Table 6) with primersspecific for the gene of interest. Oligonucleotide sequences as primerswere designed for eight antigen candidates using the public programPrimer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www slow.cgi)or picked manually. Oligonucleotide sequences as primers for PCR weredesigned for the selected antigens in order to be able to amplify thefull gene. Genomic DNA of all Klebsiella sp. strains was prepared asdescribed above. PCR was performed in a reaction volume of 30 μl usingTaq polymerase (1 U), 200 nM dNTPs, 10 pMol of each oligonucleotide,approx. 10 to 20 ng DNA and a kit according to the manufacturersinstructions (Invitrogen, The Netherlands). As standard, 30 cycles (1×:5min. 95° C., 30×:30 sec. 95° C., 30 sec. 52° C., 90 sec. 72° C., 1×:4min. 72° C.) were performed, unless conditions had to be adapted forindividual primer pairs. The PCR amplification was performed in aBiometra T3 Thermocycler. All negative PCR reactions in the firstamplification round were repeated by applying optimized conditions. TheDNA fragments were subsequently visualized by electrophoresis on a 1%agarose gel and stained with EtBr.

Sequence Analyses of Klebsiella sp. Genes

In order to determine the sequence of an antigen from diverse Klebsiellasp. strains, PCR was performed with primers specific for the gene ofinterest, as described above. Klebsiella sp. strains used for theseanalyses are shown in Table 6. Sequencing was performed with dedicatedprimers using the PCR products as templates. The sequences of theoligonucleotides are listed in Table 7. Genomic DNA of all Klebsiellasp. strains was prepared as described above. PCR was performed in areaction volume of 30 μA, as described above, unless conditions had tobe adapted for individual primer pairs. PCR samples were sequenced withthe oligonucleotides as listed in Table 7. Sequencing was performed atAgowa (Berlin, Germany).

Results

The Selected Klebsiella pneumoniae Antigens are Highly Conserved

The PCR and sequencing of the 8 selected genes were performed asdescribed under Methods. Table 6 shows the strains used for sequencing,while Table 7 lists the oligonucleotides employed for the PCR andsequencing analyses. Seven of the eight genes display a level ofsequence identity larger than app. 93% in all analyzed strains, with theexception of KPORF-21 that is at least to a level of 83% identical. Thedetailed analyses of the individual genes are described separatelybelow.

Sequence Analyses of KPORF-13

Sequences were obtained from 39 strains. The level of amino acidsequence identity ranged from 95.8% to 100% as compared to the referencesequence of KPORF-13 from Klebsiella pneumoniae MGH78578. Table 8 listsall 43 amino acid positions which showed a distinct amino acid ascompared to KPORF-13 from Klebsiella pneumoniae MGH78578.

Sequence Analyses of KPORF-21

Sequences were obtained from 49 strains. The level of amino acidsequence identity ranged from 83.3% to 100% as compared to the sequenceof KPORF-21 from Klebsiella pneumoniae MGH78578. Table 9 lists all 43amino acid positions which showed a distinct amino acid as compared toKPORF-21 from Klebsiella pneumoniae MGH78578.

Sequence Analyses of KPORF-32

Sequences were obtained from 40 strains. The level of amino acidsequence identity ranged from 92.7% to 99.5% as compared to the sequenceof KPORF-32 from Klebsiella pneumoniae MGH78578. Table 10 lists all 69amino acid positions which showed a distinct amino acid as compared toKPORF-32 from Klebsiella pneumoniae MGH78578.

Sequence Analyses of KPORF-37

Sequences were obtained from 39 strains. The level of amino acidsequence identity ranged from 99.4% to 100% as compared to the sequenceof KPORF-37 from Klebsiella pneumoniae MGH78578. Table 11 lists all 20amino acid positions which showed a distinct amino acid as compared toKPORF-37 from Klebsiella pneumoniae MGH78578.

Sequence Analyses of KPORF-38

Partial sequences were obtained from 38 strains, as the nucleotidesequence encoding the very C-terminus was not successfully amplified sofar (Table 12). The level of amino acid sequence identity ranged from98.2% to 99.9% as compared to the sequence of KPORF-38 from Klebsiellapneumoniae MGH78578. Except for partial KPORF-38 sequences obtained fromKlebsiella sp. strain i252/94 and Klebsiella sp. strain 708, allanalyzed sequences comprise the corresponding amino acid sequenceencompassing the KPORF-38.2 (amino acid range 582-1099) fragment ofKPORF-38 from Klebsiella pneumoniae MGH78578.

Sequence Analyses of KPORF-39

Sequences were obtained from 50 strains. The level of amino acidsequence identity ranged from 93.1% to 100% as compared to the sequenceof KPORF-39 from Klebsiella pneumoniae MGH78578. Table 13 lists all 29amino acid positions which showed a distinct amino acid as compared toKPORF-39 from Klebsiella pneumoniae MGH78578.

Sequence Analyses of KPORF-60

Sequences were obtained from 50 strains. The level of amino acidsequence identity ranged from 94.4% to 100% as compared to the sequenceof KPORF-60 from Klebsiella pneumoniae MGH78578. Table 14 lists all 14amino acid positions which showed a distinct amino acid as compared toKPORF-60 from Klebsiella pneumoniae MGH78578.

Sequence Analyses of KPORF-65

Sequences were obtained from 48 strains. The level of amino acidsequence identity ranged from 98.3% to 100% as compared to the sequenceof KPORF-65 from Klebsiella pneumoniae MGH78578. Table 15 lists all 27amino acid positions which showed a distinct amino acid as compared toKPORF-65 from Klebsiella pneumoniae MGH78578.

REFERENCES

The following references which have been recited in the presentspecification in a truncated version are incorporated herein byreference in their entirety.

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The features of the present invention disclosed in the specification,the claims and/or the drawings may both separately and in anycombination thereof be material for realizing the invention in variousforms thereof.

1. An isolated nucleic acid molecule encoding an antigen or a fragmentthereof, comprising a nucleic acid sequence, which is selected from thegroup consisting of: a) a nucleic acid molecule having at least 70%sequence identity to a nucleic acid molecule having a nucleotidesequence selected from the group comprising Seq ID Nos: 1 to 187 and SeqID No 375, b) a nucleic acid molecule which is complementary to thenucleic acid molecule of a), c) a nucleic acid molecule comprising atleast 15 sequential bases of the nucleic acid molecule of a) or b), d) anucleic acid molecule which anneals under stringent hybridisationconditions to the nucleic acid molecule of a), b), or c), e) a nucleicacid molecule which, but for the degeneracy of the genetic code, wouldhybridize to the nucleic acid molecule defined in a), b), c), or d). 2.The isolated nucleic acid molecule according to claim 1, wherein thesequence identity to Seq ID Nos: 1 to 187 or Seq ID No 375 is at least80%.
 3. The nucleic acid molecule according to claim 1, wherein thenucleic acid is DNA or RNA.
 4. (canceled)
 5. An isolated nucleic acidmolecule according to claim 1, wherein the nucleic acid molecule isisolated from a genomic DNA from a Klebsiella species.
 6. The nucleicacid according to claim 1, wherein the fragment is an active fragment oran active variant thereof.
 7. The nucleic acid according to claim 1,wherein the antigen or fragment thereof, comprises or consists of apolypeptide or peptide fragment from Klebsiella.
 8. A vector comprisinga nucleic acid molecule according to claim
 1. 9. (canceled)
 10. A hostcell comprising the vector as defined in claim
 8. 11. An antigen that isimmunologically reactive with sera from a human having a Klebsiellainfection, or from an uninfected healthy human who was previouslyinfected with Klebsiella.
 12. An antigen comprising or consisting of anisolated polypeptide selected from the group consisting of Seq ID Nos:188 to 374, Seq ID No 376 or an active fragment or an active variantthereof.
 13. The antigen according to claim 12, wherein the polypeptideis encoded by a nucleic acid molecule as defined in claim
 1. 14. Theantigen according to claim 12, wherein the active fragment thereofconsists of at least 50%, of said polypeptide.
 15. The antigen accordingto claim 12, wherein the active variant thereof has at least 50%sequence identity to said polypeptide.
 16. The antigen according toclaim 14, wherein the active fragment thereof comprises or consists ofan amino acid sequence selected from the group consisting of amino acids2-130 of Seq ID No 205; amino acids 26-356 of Seq ID No 216; amino acids2-180 of Seq ID No 223; amino acids 1-168 of Seq ID No 224; amino acids23-397 of Seq ID No 235; amino acids 2-420 and 414-847 of Seq ID No 240;amino acids 582-1099 of Seq ID No 241; amino acids 1-245 of Seq ID No242; amino acids 24-703 of Seq ID No 247; amino acids 23-328 of Seq IDNo 252; amino acids 23-248 of Seq ID No 263; amino acids 2-335 of Seq IDNo 267; amino acids 38-633 of Seq ID No 268; amino acids 26-742 of SeqID No 269; amino acids 26-429 of Seq ID No 281; and amino acids 1-632 ofSeq ID No
 285. 17. The antigen according to claim 15, wherein the activevariant thereof has at least 50% sequence identity to an amino acidsequence selected from the group consisting of amino acids 2-130 of SeqID No 205; amino acids 26-356 of Seq ID No 216; amino acids 2-180 of SeqID No 223; amino acids 1-168 of Seq ID No 224; amino acids 23-397 of SeqID No 235; amino acids 2-420 and 414-847 of Seq ID No 240; amino acids582-1099 of Seq ID No 241; amino acids 1-245 of Seq ID No 242; aminoacids 24-703 of Seq ID No 247; amino acids 23-328 of Seq ID No 252;amino acids 23-248 of Seq ID No 263; amino acids 2-335 of Seq ID No 267;amino acids 38-633 of Seq ID No 268; amino acids 26-742 of Seq ID No269; amino acids 26-429 of Seq ID No 281; and amino acids 1-632 of SeqID No
 285. 18. The antigen according to claim 12, wherein the activevariant thereof is derived from the homologous sequence of a differentserotype of K. pneumoniae.
 19. The antigen according to claim 12,wherein the active variant is selected from the group consisting of SEQID No 413 to
 765. 20. The antigen according to claim 16 or 17, wherebythe antigen is further defined by a) 1 to 400 additional amino acidresidue(s) to the active fragment of the antigen comprising orconsisting of amino acids 2-420 or 414-847 of Seq ID No 240 or to theactive variant of the antigen derived from amino acids 2-420 or 414-847of Seq ID No 240; or b) 1 to 1100 additional amino acid residue(s) tothe active fragment of the antigen comprising or consisting of aminoacids 582-1099 of Seq ID No 241 or to the active variant of the antigenderived from amino acids 582-1099 of Seq ID No
 241. 21-22. (canceled)23. The antigen according to claim 20, whereby said additional aminoacid residue(s) is/are flanking the antigen defined by a) amino acids2-420 of Seq ID No 240 or the variant derived thereof C-terminally, b)amino acids 414-847 of Seq ID No 240 or the variant derived thereofN-terminally, or c) amino acids 582-1099 of Seq ID No 241 or the variantderived thereof N- and/or C-terminally.
 24. (canceled)
 25. An antigencomprising at least one core amino acid sequence as indicated in column“Predicted immunogenic aa” or “Location of identified immunogenicregion” of Table 1, or as defined by columns “From aa” and “To aa” ofTable 4, or as indicated in column “Location in protein (aa)” of Table5, whereby more preferably the core amino acid sequence is selected fromthe group consisting of: amino acids 11-27, 35-47, 68-107, 113-122,124-136, 140-146, 152-164, 168-174, 183-201, 211-218, 228-243, 246-253and 180-226 of Seq ID No 204; amino acids 13-31, 48-59, 69-91, 109-115,121-127 and 46-105 of Seq ID No 205; amino acids 12-44, 49-95, 102-145,148-178, 184-229, 233-244, 249-273, 292-299, 304-329, 334-348, 354-365,367-385, 394-426, 428-440, 444-487, 503-527, 531-539, 546-554, 556-584and 273-286 of Seq ID No 206; amino acids 7-17, 22-32, 34-41, 55-77,79-86, 93-111, 118-126, 131-148, 152-162, 165-177, 183-197, 213-220,234-250, 253-262, 267-294 and 211-269 of Seq ID No 207; amino acids22-29, 41-56, 58-66, 79-88, 94-121, 124-131, 134-157, 162-171, 173-180,189-197, 201-214, 216-224, 242-254, 257-270, 282-287, 290-302, 309-315,320-325, 341-355, 362-368, 372-378 and 1-48 of Seq ID No 208; aminoacids 5-15, 18-35, 48-61, 65-71, 112-119, 138-154, 157-169, 179-208,214-223, 226-232, 243-250, 256-262, 277-286, 289-296, 338-348, 352-363,370-376, 385-408, 420-436, 443-454, 462-483, 498-561, 563-592, 600-642,661-671, 673-709, 714-733, 748-754, 771-776, 798-806, 808-821, 823-839and 31-83 of Seq ID No 209; amino acids 5-14, 21-26, 31-41, 59-77,101-115, 132-145, 147-156, 180-185, 188-197 and 97-158 of Seq ID No 210;amino acids 6-18, 23-43, 45-56, 69-80, 87-97, 112-123, 135-151, 164-171,178-193, 200-227, 249-258, 262-274, 279-291, 302-308, 322-327, 329-336,351-363, 366-373, 384-399, 403-411, 415-434, 440-446, 461-482, 488-506,510-516, 518-551, 574-589, 607-629, 634-665, 667-687, 694-712, 725-739,743-751, 753-768 and 521-583 of Seq ID No 211; amino acids 4-13, 19-44,55-63, 71-82, 89-110, 120-130, 132-138, 145-161, 168-182, 189-258,261-272, 278-288, 290-301 and 11-76 of Seq ID No 212; amino acids 4-22,43-56, 63-68, 81-90, 93-99, 139-148, 155-160, 170-176, 189-195, 207-218,227-232, 241-249, 251-258, 260-266, 277-295, 300-327, 329-336, 340-356,384-390, 418-423, 427-433, 438-444 and 383-428 of Seq ID No 213; aminoacids 10-18, 32-37, 45-55, 60-69, 77-83, 89-95, 120-125, 133-170,172-185, 193-211, 214-223, 232-249, 255-275, 277-303, 305-310, 320-328,334-341, 347-353, 355-369, 380-386, 389-395 and 71-85 of Seq ID No 214;amino acids 4-23, 27-35, 67-73, 80-103, 117-126, 132-138, 140-159,162-171, 180-194, 198-208, 211-218, 228-234, 239-253, 262-270, 272-291,296-305 and 39-110 of Seq ID No 215; amino acids 13-24, 27-34, 37-66,69-88, 99-104, 149-155, 164-175, 184-193, 199-209, 227-235, 264-273,276-285, 288-315, 323-335, 346-353, 56-111 and 199-261 of Seq ID No 216;amino acids 11-22, 25-48, 51-60, 64-72, 80-96, 108-122, 132-137,142-150, 152-167, 175-199, 214-229, 237-244, 252-258, 260-266, 279-287,301-340, 345-350 and 109-153 of Seq ID No 217; amino acids 37-43, 50-57,65-82, 87-109, 123-129, 141-150, 152-157, 166-172, 179-203, 209-241,249-284, 290-300, 308-326, 329-335, 345-357, 359-368, 379-386, 390-417,420-425, 438-444, 461-466, 473-490, 497-505, 524-534, 541-550, 586-597,608-614, 622-632, 660-666, 679-694, 696-706, 708-722, 725-731, 737-763,784-789, 810-825, 837-854, 857-880, 882-895, 901-907, 911-928, 14-76 and176-220 of Seq ID No 218; amino acids 9-16, 38-52, 61-86, 93-100,110-117, 123-132, 138-145, 151-169, 172-181, 186-202, 208-225, 227-253,264-275, 289-295, 320-329, 335-342 and 113-193 of Seq ID No 219; aminoacids 11-18, 24-30, 42-49, 53-63, 69-80, 87-93, 95-103, 144-171,173-185, 193-200, 202-208, 215-221, 242-261, 266-273, 277-286, 290-299,322-328, 338-351, 354-377, 391-409, 441-451, 461-466, 499-515, 521-527,562-569, 621-629, 647-663, 676-682, 694-701, 703-713, 725-731, 735-744,755-764, 793-800 and 490-547 of Seq ID No 220; amino acids 4-11, 14-22,38-70, 81-90, 97-114, 118-132, 147-171, 173-181, 187-202, 244-250,252-298, 301-311, 313-331, 342-368, 410-418, 446-451, 456-462, 468-474,476-492, 499-507, 519-528, 552-565, 568-575, 584-613, 618-624, 626-649and 417-489 of Seq ID No 221; amino acids 4-9, 32-53, 66-72, 74-90,97-104, 110-130, 133-139, 144-152, 166-177, 203-213, 215-241, 256-275,291-304, 307-316, 321-326, 334-345, 352-367 and 201-255 of Seq ID No222; amino acids 13-19, 26-43, 66-72, 80-85, 95-101, 109-125, 131-137and 25-107 of Seq ID No 223; amino acids 13-24, 35-43, 50-56, 58-68,77-83, 104-110, 117-125, 132-138, 140-153 and 19-66 of Seq ID No 224;amino acids 15-31, 37-42, 47-54, 68-87, 89-96, 107-117, 121-127,131-137, 145-151, 176-182, 220-226, 232-246, 250-257, 291-300, 317-325,328-333, 337-359, 368-393, 403-428, 460-478, 480-493, 500-506, 511-516,519-526, 528-559, 565-572, 584-595, 597-605, 608-613, 626-648, 679-684,687-693, 703-714, 718-735, 742-750, 757-765, 768-788, 793-799, 813-819,823-829, 839-850 and 576-623 of Seq ID No 225; amino acids 10-35, 37-60,63-76, 79-86, 88-97, 108-113, 118-126, 128-134, 138-145, 153-159,168-188, 194-208, 211-243, 255-260, 270-276, 285-301, 307-346, 348-367and 275-339 of Seq ID No 226; amino acids 4-17, 21-33, 35-42, 47-64,72-80, 85-92, 98-103, 125-147, 151-161, 165-177, 183-230, 232-246,256-262, 284-306, 310-328, 331-367, 369-383, 392-399 and 32-85 of Seq IDNo 227; amino acids 5-11, 18-27, 42-52, 60-65, 75-84, 90-102, 107-116,125-178, 184-206, 221-233, 235-242, 249-257, 264-277, 288-317 and267-313 of Seq ID No 228; amino acids 5-11, 14-42, 50-75, 79-86, 89-98,120-125, 152-160, 166-181, 185-193, 200-207 and 85-114 of Seq ID No 229;amino acids 4-30, 36-43, 46-55, 63-111, 144-152, 159-168, 179-189,191-200, 205-213 and 37-109 of Seq ID No 230; amino acids 20-45, 57-77,80-100, 119-126, 131-137, 143-169, 179-185, 195-203, 207-231, 235-264,282-302, 320-329, 341-347, 353-359, 361-373 and 266-296 of Seq ID No231; amino acids 5-22, 24-37, 41-55, 57-65, 72-78, 90-103, 105-116,119-130, 164-170, 190-202, 209-231, 244-254, 260-276, 300-339, 344-350,355-376, 389-397, 399-406, 408-421, 429-437 and 103-152 of Seq ID No232; amino acids 8-16, 18-25, 31-47, 71-82, 87-102, 104-114, 126-156,176-183, 190-200, 205-212, 218-228, 231-243, 256-279, 287-301, 303-312,324-332, 335-348, 351-357, 365-380, 395-412, 422-451, 456-464, 467-483,501-507 and 405-468 of Seq ID No 233; amino acids 4-18, 21-39, 46-56,63-69, 72-86, 116-130, 132-160, 162-190, 196-201, 209-231, 233-241,251-265, 269-282, 292-298, 309-324, 333-369, 391-415, 417-427, 436-454,471-480, 482-499, 510-518, 521-533, 537-543, 545-561, 571-581, 585-597,599-607, 609-635, 638-643, 650-665, 671-685, 687-695, 701-707, 710-720,724-736, 747-757, 764-769, 772-784, 791-796, 808-820 and 317-401 of SeqID No 234; amino acids 4-12, 15-33, 58-77, 82-89, 98-106, 108-118,120-135, 141-147, 152-160, 168-215, 225-233, 235-247, 250-264, 284-312,314-321, 336-343, 359-374, 386-394 and 159-218 of Seq ID No 235; aminoacids 4-16, 24-36, 40-47, 49-56, 61-81, 84-143, 148-156, 158-164,170-175, 194-206, 208-214 and 126-203 of Seq ID No 236; amino acids28-45, 50-61, 94-111, 113-124, 137-142, 147-173, 180-188, 190-196,202-223, 229-235, 239-249, 262-270, 280-288, 290-321, 325-332, 347-355,359-368, 389-407, 415-427, 429-453, 458-465, 477-485, 499-505, 516-527,531-549, 569-592, 594-602, 605-615, 628-635, 647-659, 662-683, 727-735,760-765, 771-780, 788-809, 811-818 and 549-630 of Seq ID No 237; aminoacids 21-28, 33-40, 48-100, 104-111, 113-134 and 1-46 of Seq ID No 238;amino acids 12-24, 31-41, 53-61, 73-87, 112-128, 133-140, 151-156 and26-98 of Seq ID No 239; amino acids 4-9, 19-26, 32-56, 58-67, 71-81,90-95, 97-105, 112-118, 124-132, 138-144, 147-167, 169-177, 199-207,212-217, 231-241, 250-260, 266-272, 274-282, 289-296, 299-310, 316-331,344-350, 352-363, 368-377, 381-394, 399-406, 412-450, 459-473, 486-503,508-514, 518-548, 564-570, 579-587, 602-608, 616-623, 628-635, 638-654,678-688, 691-696, 703-709, 716-723, 761-772, 784-793, 819-826, 835-844and 790-834 of Seq ID No 240; amino acids 4-10, 18-36, 43-50, 63-71,75-105, 109-117, 134-140, 145-157, 176-182, 184-201, 203-211, 215-225,240-250, 262-284, 294-309, 313-319, 327-337, 350-356, 361-367, 372-393,411-421, 428-451, 453-466, 487-492, 501-528, 535-553, 564-574, 592-605,612-629, 631-640, 646-653, 658-666, 673-681, 713-718, 720-730, 739-749,784-792, 821-826, 833-844, 853-863, 871-876, 885-894, 900-918, 937-950,952-957, 972-990, 995-1001, 1024-1036, 1039-1044, 1049-1055, 1062-1089,1091-1103, 1110-1121, 1123-1129, 1131-1151, 1157-1179, 1181-1201,1204-1223, 1233-1244, 1269-1276, 1279-1286, 1294-1301, 1303-1309,1315-1338, 1350-1362, 1373-1381, 1398-1406, 1412-1423, 1440-1446,1458-1466, 1481-1487, 1492-1508, 1511-1518, 1528-1534, 1536-1547,1553-1565, 1606-1617, 1619-1644 and 761-781 of Seq ID No 241; aminoacids 6-13, 31-38, 47-60, 71-102, 107-123, 128-155, 173-179, 185-194,210-220 and 161-232 of Seq ID No 242; amino acids 11-34, 36-43, 49-67,74-79, 84-92, 94-100, 103-112, 120-129, 134-155, 162-173, 177-185,189-202, 206-211 and 130-185 of Seq ID No 243; amino acids 4-10, 20-35,37-46, 48-55, 60-66, 75-82, 87-98, 133-150, 166-172, 178-189, 208-214,230-235, 245-251, 271-308, 319-333, 335-355, 373-380 and 117-201 of SeqID No 244; amino acids 4-30, 54-65, 91-105, 107-131, 135-154, 163-192,199-208, 210-224, 229-239, 248-257, 263-279, 281-294, 328-354, 373-379,382-405, 426-453, 462-487 and 249-323 of Seq ID No 245; amino acids4-10, 12-24, 45-55, 75-88 and 24-40 of Seq ID No 246; amino acids 4-14,20-37, 47-53, 55-61, 75-81, 97-103, 107-124, 129-135, 139-147, 160-166,169-175, 181-190, 202-221, 247-255, 272-285, 300-310, 318-332, 351-361,384-397, 406-427, 442-449, 458-482, 494-503, 512-524, 531-539, 552-562,577-588, 590-596, 600-608, 613-624, 637-668, 692-700 and 232-278 of SeqID No 247; amino acids 33-39, 49-55, 68-84, 90-96, 104-120, 126-143,150-159, 168-191, 197-208, 219-225, 227-233, 241-247, 63-115 and 200-250of Seq ID No 248; amino acids 4-22, 24-34, 36-55, 57-76, 83-97, 99-117,135-143, 145-157, 163-174, 178-198, 200-207, 209-270, 276-290, 321-335,338-347, 367-374, 393-402, 404-411, 416-422, 443-460, 467-473 and117-183 of Seq ID No 249; amino acids 26-37, 44-52, 57-96, 104-111,118-124, 155-177, 179-197, 201-214, 223-233, 243-250, 257-262, 291-297,303-314, 319-363 and 47-105 of Seq ID No 250; amino acids 36-43, 45-60,76-97, 107-125, 131-156, 158-164 and 118-163 of Seq ID No 251; aminoacids 5-32, 40-50, 52-60, 70-88, 92-101, 106-126, 138-150, 152-161,175-193, 201-234, 237-248, 270-285, 297-303, 312-318 and 209-255 of SeqID No 252; amino acids 4-12, 23-34, 49-55, 59-65, 70-81, 83-130 and62-113 of Seq ID No 253; amino acids 4-26, 38-49, 69-76, 82-96, 103-119,126-140, 143-190, 194-209, 212-218 and 100-167 of Seq ID No 254; aminoacids 7-29, 35-47, 56-66, 80-94, 97-123, 125-148, 150-160, 166-173,175-191, 193-200, 207-225 and 75-176 of Seq ID No 255; amino acids14-36, 39-45, 51-59, 66-71, 76-88, 106-117, 121-126, 140-157, 164-187,198-206, 210-252 and 202-256 of Seq ID No 256; amino acids 4-19, 27-35,90-107, 120-134, 144-150, 166-175, 192-198, 221-243, 249-255, 263-278,283-288, 305-321, 324-334, 342-349, 355-366, 377-390, 413-425, 442-448and 130-178 of Seq ID No 257; amino acids 17-26, 41-51, 54-61, 64-72,78-105, 117-125, 127-137, 147-155, 175-213, 230-236, 238-261, 271-277,282-297, 309-318, 329-347, 355-372, 377-390 and 69-126 of Seq ID No 258;amino acids 4-48, 54-60, 62-69, 73-81, 88-115, 124-137, 139-154,156-169, 171-190, 194-231, 240-273, 288-303, 336-363, 367-395, 405-411,434-442, 449-454, 466-483, 491-507 and 226-282 of Seq ID No 259; aminoacids 26-34, 39-47, 50-80, 82-88, 97-105, 108-127, 131-137, 162-180,185-191, 198-203, 209-214, 226-247, 256-288, 296-305 and 149-239 of SeqID No 260; amino acids 5-28, 30-54, 73-84, 89-98, 109-116, 122-128,137-142, 163-189, 207-236, 245-280, 288-390, 404-423, 426-433, 450-474,487-504, 506-513, 524-530, 532-595, 605-614, 620-626, 631-638, 644-657,667-683, 686-693, 695-702, 707-733, 739-747 and 6-62 of Seq ID No 261;amino acids 23-31, 39-50, 55-67, 76-100, 117-130, 149-171, 173-185,218-238, 242-288, 291-298, 334-346, 355-369, 382-399, 413-420, 431-438,442-449, 455-466, 486-493, 498-508, 524-531, 540-546, 551-558, 562-570,575-582, 585-596, 598-604, 621-630, 632-650, 670-677, 682-701, 736-749,755-761 and 612-626 of Seq ID No 262; amino acids 4-21, 24-39, 44-68,74-81, 85-91, 109-116, 129-138, 142-148, 173-188, 195-201, 207-212,223-228 and 126-148 of Seq ID No 263; amino acids 4-17, 24-42, 61-67,84-93, 96-102, 116-121, 135-143, 155-165, 177-186, 210-224, 253-259,272-297, 299-331, 337-351, 359-367, 369-385 and 1-49 of Seq ID No 264;amino acids 4-25, 28-54, 67-81, 85-136, 138-143, 157-170, 180-190,197-203, 205-214, 219-243, 246-270, 277-283, 290-299, 305-311 and127-182 of Seq ID No 265; amino acids 11-20, 25-33, 75-80, 85-91,113-124, 143-155, 161-170, 172-184 and 128-176 of Seq ID No 266; aminoacids 4-9, 16-26, 28-34, 55-80, 120-143, 150-156, 158-164, 167-178,185-190, 192-213, 221-237, 242-255, 257-272, 281-290, 325-332 and 48-106of Seq ID No 267; amino acids 13-48, 59-70, 78-88, 95-112, 129-151,153-161, 163-182, 214-221, 235-245, 248-277, 281-291, 293-301, 303-311,315-320, 323-346, 377-383, 390-398, 447-454, 474-487, 491-512, 531-544,547-553, 582-590, 597-603, 605-611, 623-629 and 410-466 of Seq ID No268; amino acids 6-26, 39-46, 48-58, 69-75, 109-121, 139-144, 148-155,166-172, 215-221, 261-267, 313-319, 363-386, 423-433, 447-458, 465-471,483-494, 497-517, 558-565, 578-586, 589-597, 619-626, 636-645, 659-665,671-680, 682-693, 733-739 and 152-206 of Seq ID No 269; amino acids4-19, 23-35, 40-50, 52-58, 65-73, 78-103, 112-125, 146-160, 163-192,194-200 and 29-90 of Seq ID No 270; amino acids 4-13, 17-32, 40-50,57-67, 76-81, 88-95, 107-119, 131-142, 144-157, 171-178, 185-193,197-207, 212-227, 231-238, 248-253, 263-310 and 90-170 of Seq ID No 271;amino acids 9-28, 57-82, 84-93, 126-135, 143-166, 173-194, 196-201,212-220, 228-254, 269-277, 289-298, 305-316, 320-327, 330-337, 350-359,373-378, 386-392, 403-411, 421-428, 435-441, 443-458, 465-470 and 80-141of Seq ID No 272; amino acids 11-48, 54-67, 69-75, 89-95, 101-122,124-131, 134-157, 159-175, 202-208, 214-228, 258-270, 272-280, 287-295,298-310, 331-338, 340-417, 427-500, 502-509, 534-552, 556-561, 564-577,585-592, 594-608, 621-627, 632-641, 643-652, 671-681, 683-709, 712-743,758-764, 776-783, 789-820, 835-851, 864-883, 885-910, 913-940, 948-953,967-976, 994-1020 and 775-825 of Seq ID No 273; amino acids 14-24,32-54, 58-63, 70-80, 93-100, 108-125, 127-135, 142-153, 155-160,180-191, 201-208, 210-216, 222-235, 242-264, 267-273, 276-282, 284-308and 10-59 of Seq ID No 274; amino acids 16-28, 44-68, 70-77, 83-90,99-129, 131-137, 145-154, 161-175, 183-190, 196-203, 205-220, 238-245,321-328, 330-338, 366-379, 383-397, 399-405, 412-418, 442-458, 471-483,486-505, 536-544, 562-568, 583-602, 610-618, 629-635, 641-655, 672-682,697-705, 714-729, 744-751, 755-762, 766-771, 783-807 and 555-621 of SeqID No 275; amino acids 4-9, 20-34, 45-54, 60-77, 79-89, 91-100, 102-149,162-170, 177-189, 193-208, 210-222, 238-244, 252-264, 267-276, 302-307and 100-140 of Seq ID No 276; amino acids 11-27, 30-49, 56-62, 69-74,76-85, 94-108, 116-125, 129-147, 153-161, 165-171, 177-208, 217-223,225-231, 237-255, 260-284, 293-300 and 73-137 of Seq ID No 277; aminoacids 4-38, 40-51, 84-97, 99-106, 109-115, 119-129, 131-145, 148-160,180-186, 188-202, 230-243, 246-267, 274-288, 290-299, 302-312, 317-327,332-344, 353-377, 381-388, 407-419, 423-437, 447-470, 474-482, 486-494,501-523, 531-546, 551-556 and 727-740 of Seq ID No 278; amino acids23-52, 62-76, 87-104, 109-115, 117-123, 129-139, 143-149, 152-170,172-191, 199-205, 212-218, 220-240, 249-256, 263-275, 297-303, 308-342,349-380, 382-394, 414-420, 430-441, 446-452, 460-475, 488-505, 514-531,533-539, 546-568, 570-577, 579-588, 613-625, 632-670, 672-716, 718-745,759-769, 785-798, 801-807 and 272-324 of Seq ID No 279; amino acids4-34, 36-43, 56-73, 80-87, 101-134, 148-159, 161-170, 178-185, 195-206,211-221, 223-248, 259-271, 276-295, 297-308 and 241-296 of Seq ID No280; amino acids 5-31, 44-50, 64-74, 86-94, 132-147, 154-167, 196-203,209-219, 253-260, 284-289, 300-312, 319-327, 335-340, 358-364, 376-383and 166-202 of Seq ID No 281; amino acids 4-9, 12-27, 29-71, 77-84,90-108, 114-142, 147-164, 180-213, 217-227, 229-282, 291-309, 322-329,336-353, 365-370 and 317-364 of Seq ID No 282; amino acids 36-41, 52-66,71-83, 89-95, 116-127, 154-174, 176-184, 200-206, 230-237, 248-259,269-284, 307-316, 376-383, 399-418, 424-442, 445-451, 454-462 and 1-50of Seq ID No 283; amino acids 9-14, 33-49, 64-72, 87-92, 103-109,123-128, 130-141, 143-154, 160-166, 182-214, 237-247, 251-260, 292-300,327-332, 337-350, 357-365, 388-398, 405-411, 422-428, 451-459, 478-488,520-531, 534-540, 558-564, 580-586, 591-600, 605-615, 629-635, 641-653,658-672, 212-244 and 533-611 of Seq ID No 284; amino acids 4-10, 17-27,30-37, 44-62, 80-85, 94-114, 118-131, 134-141, 148-161, 171-212,218-241, 248-261, 274-313, 325-336, 342-348, 359-373, 391-397, 424-431,454-474, 489-495, 497-503, 505-515, 548-553, 560-580, 591-610 and277-324 of Seq ID No 285; amino acids 7-16, 18-24, 30-47, 49-70, 83-99,103-117, 126-141, 146-153, 159-165, 177-194, 198-221, 236-246, 255-262,273-279, 283-296, 301-332, 338-411, 422-428, 434-440, 452-458, 463-469,494-509, 511-517, 524-531, 548-554, 564-572 and 335-389 of Seq ID No286; amino acids 9-15, 33-54, 56-80, 102-108 and 1-42 of Seq ID No 287;amino acids 15-36, 42-55, 58-68 and 54-77 of Seq ID No 288; amino acids55-75, 89-96, 98-110 and 14-36 of Seq ID No 289; amino acids 8-14,29-51, 73-101, 110-117 and 70-114 of Seq ID No 290; amino acids 20-25,29-34, 41-52, 60-67, 69-85, 90-100, 114-122, 136-142, 160-170, 174-181and 21-58 of Seq ID No 291; amino acids 14-22 and 4-13 of Seq ID No 292;amino acids 22-40, 54-66, 88-105, 109-118 and 31-74 of Seq ID No 293;amino acids 5-11, 18-32, 47-60, 66-73, 83-92, 113-120, 126-141, 151-164,167-174, 201-211 and 118-129 of Seq ID No 294; amino acids 5-11, 18-24,32-40, 47-53 and 25-54 of Seq ID No 295; amino acids 18-24, 31-48 and5-55 of Seq ID No 296; amino acids 10-16, 26-32, 47-56, 85-95 and 10-62of Seq ID No 297; amino acids 4-12, 16-26 and 25-34 of Seq ID No 298;amino acids 19-29, 45-51, 63-68, 76-92, 103-110, 114-120, 123-133,135-141 and 14-78 of Seq ID No 299; amino acids 4-18, 47-61 and 57-93 ofSeq ID No 300; amino acids 17-29, 44-50 and 26-38 of Seq ID No 301;amino acids 5-19, 55-64, 78-85, 95-101, 104-112 and 24-33 of Seq ID No302; amino acids 4-10 and 12-31 of Seq ID No 303; amino acids 4-12,27-41, 43-58, 60-67, 76-86 and 13-65 of Seq ID No 304; amino acids30-38, 57-67 and 5-32 of Seq ID No 305; amino acids 30-43 and 2-21 ofSeq ID No 306; amino acids 14-20, 23-36, 41-48 and 1-52 of Seq ID No307; amino acids 18-33, 51-58, 76-82 and 32-46 of Seq ID No 308; aminoacids 25-31 and 2-16 of Seq ID No 309; amino acids 14-23, 50-58 and 9-49of Seq ID No 310; amino acids 4-10, 22-31, 35-45, 48-68, 71-80 and 17-66of Seq ID No 311; amino acids 4-24, 28-42, 46-56, 63-69, 87-94, 112-131and 2-46 of Seq ID No 312; amino acids 4-15, 19-28, 34-41, 52-62, 78-86and 2-20 of Seq ID No 313; amino acids 4-11, 16-30, 32-42 and 7-38 ofSeq ID No 314; amino acids 4-20, 22-31 and 22-38 of Seq ID No 315; aminoacids 4-19 and 17-32 of Seq ID No 316; amino acids 7-13, 17-22, 27-33,80-100 and 26-40 of Seq ID No 317; amino acids 10-18, 22-48 and 32-44 ofSeq ID No 318; amino acids 15-24, 43-49, 73-83 and 45-93 of Seq ID No319; amino acids 22-29, 46-55, 57-63 and 5-17 of Seq ID No 320; aminoacids 10-33 and 21-35 of Seq ID No 321; amino acids 16-24 and 22-49 ofSeq ID No 322; amino acids 4-16, 37-73, 76-110, 117-125, 127-132 and2-30 of Seq ID No 323; amino acids 4-12, 23-35, 44-56, 59-88 and 22-76of Seq ID No 324; amino acids 15-26 and 23-35 of Seq ID No 325; aminoacids 12-22, 31-40 and 17-44 of Seq ID No 326; amino acids 4-9, 13-18,29-35 and 57-64 of Seq ID No 327; amino acids 31-55, 67-81 and 25-70 ofSeq ID No 328; amino acids 13-24, 51-58 and 13-26 of Seq ID No 329;amino acids 6-20, 29-40, 57-79 and 46-88 of Seq ID No 330; amino acids8-14, 41-54, 68-76, 83-93, 106-126, 130-139 and 12-72 of Seq ID No 331;amino acids 5-13, 17-24, 41-55, 64-69, 80-85, 94-107, 109-115 and 53-88of Seq ID No 332; amino acids 5-12, 32-54, 57-64 and 20-33 of Seq ID No333; amino acids 4-16, 40-48, 50-58, 62-68, 75-85, 92-104, 108-116,124-134 and 68-128 of Seq ID No 334; amino acids 7-13, 19-29, 34-40,54-71, 76-81, 91-144, 147-155, 157-188 and 11-83 of Seq ID No 335; aminoacids 17-24, 32-41 and 6-43 of Seq ID No 336; amino acids 14-31, 38-59,69-87, 95-102, 126-146, 157-162, 177-193, 201-227, 238-251 and 63-78 ofSeq ID No 337; amino acids 10-16, 18-25, 27-41, 43-52, 59-86, 94-101,134-140 and 38-100 of Seq ID No 338; amino acids 4-19, 23-35, 43-72,78-92 and 37-93 of Seq ID No 339; amino acids 15-20, 27-32, 41-65,69-82, 93-105, 107-115, 120-147, 170-178, 184-201, 214-257, 272-281,293-314, 332-339, 358-364, 374-381, 390-397, 399-414, 428-460 and317-375 of Seq ID No 340; amino acids 11-28, 47-55, 59-68, 76-105,108-116, 120-144, 146-160, 167-175, 180-187, 209-233 and 144-158 of SeqID No 341; amino acids 4-13, 58-78 and 14-77 of Seq ID No 342; aminoacids 26-31, 44-49, 57-64, 67-74, 107-112, 116-152, 154-181, 202-212,241-255 and 57-101 of Seq ID No 343; amino acids 10-41, 53-70, 81-93,100-111, 137-147, 164-169, 183-190, 199-210, 216-221, 226-240 and 84-95of Seq ID No 344; amino acids 12-45, 48-56, 73-79, 91-103, 106-112,117-125, 132-143, 154-160, 178-201, 208-214, 216-225, 260-266, 276-283and 98-115 of Seq ID No 345; amino acids 4-15, 30-42 and 29-39 of Seq IDNo 346; amino acids 22-53, 55-73, 80-88 and 33-66 of Seq ID No 347;amino acids 6-23, 44-54 and 56-67 of Seq ID No 348; amino acids 8-21,35-44, 66-75, 82-87, 94-101 and 32-94 of Seq ID No 349; amino acids8-20, 23-32, 36-50, 53-69 and 15-69 of Seq ID No 350; amino acids 8-22of Seq ID No 351; amino acids 31-37 and 2-31 of Seq ID No 352; aminoacids 4-20, 23-39, 58-63, 71-78, 97-102 and 22-82 of Seq ID No 353;amino acids 23-44, 135-152, 168-184 and 57-116 of Seq ID No 354; aminoacids 24-31, 42-50, 52-62, 93-117 and 43-94 of Seq ID No 355; aminoacids 20-29 and 24-43 of Seq ID No 356; amino acids 12-57, 59-74 and22-40 of Seq ID No 357; amino acids 7-16, 18-26, 39-45, 68-78, 86-92 and65-82 of Seq ID No 358; amino acids 5-17, 19-34, 42-48, 56-71, 102-113,118-129 and 67-111 of Seq ID No 359; amino acids 4-33, 50-71 and 13-55of Seq ID No 360; amino acids 9-17, 23-30, 37-54, 69-88, 96-102,114-123, 130-140, 143-163 and 5-70 of Seq ID No 361; amino acids 4-23,27-52, 71-80 and 9-94 of Seq ID No 362; amino acids 13-19 and 2-21 ofSeq ID No 363; amino acids 18-26, 28-52, 63-74, 94-107, 123-134 and18-84 of Seq ID No 364; amino acids 19-33, 57-68 and 26-48 of Seq ID No365; amino acids 4-26, 31-37, 42-59 and 12-65 of Seq ID No 366; aminoacids 4-25 and 20-39 of Seq ID No 367; amino acids 40-51, 54-62, 67-75,83-89, 126-146, 148-156 and 31-42 of Seq ID No 368; amino acids 4-15,23-33, 38-49, 82-98 and 7-91 of Seq ID No 369; amino acids 6-26, 36-57and 40-64 of Seq ID No 370; amino acids 6-15, 21-28, 32-38, 57-65,78-103, 114-134, 138-144, 154-163 and 41-95 of Seq ID No 371; aminoacids 13-30, 47-57, 71-76 and 25-71 of Seq ID No 372; amino acids 4-31,43-51, 55-63, 67-72, 76-83, 88-95, 99-118, 125-132, 134-159 and 82-118of Seq ID No 373; amino acids 4-17, 26-32, 34-40, 45-61, 67-92 and 41-97of Seq ID No 374; amino acids 179-208 and 198-227 of Seq ID No 204;amino acids 45-69, 65-89 and 83-106 of Seq ID No 205; amino acids269-290 of Seq ID No 206; amino acids 209-230, 226-249 and 245-269 ofSeq ID No 207; amino acids-9-15, 10-33 and 28-52 of Seq ID No 208; aminoacids 29-50, 45-67 and 62-85 of Seq ID No 209; amino acids 96-120,115-139 and 134-158 of Seq ID No 210; amino acids 519-543, 539-563 and559-584 of Seq ID No 211; amino acids 10-35, 31-56 and 52-77 of Seq IDNo 212; amino acids 382-407 and 403-428 of Seq ID No 213; amino acids66-90 of Seq ID No 214; amino acids 38-65 and 61-88 of Seq ID No 215;amino acids 56-85, 198-221, 217-240 and 236-261 of Seq ID No 216; aminoacids 108-132 and 128-153 of Seq ID No 217; amino acids 13-37, 33-56,52-76, 175-200 and 196-220 of Seq ID No 218; amino acids 132-156,152-176 and 172-195 of Seq ID No 219; amino acids 489-512, 508-531 and526-549 of Seq ID No 220; amino acids 416-442, 438-465 and 461-489 ofSeq ID No 221; amino acids 199-222, 217-240 and 235-257 of Seq ID No222; amino acids 25-55, 51-81 and 77-107 of Seq ID No 223; amino acids18-46 and 42-66 of Seq ID No 224; amino acids 575-601 and 597-623 of SeqID No 225; amino acids 274-299, 295-320 and 316-339 of Seq ID No 226;amino acids 32-61 and 57-85 of Seq ID No 227; amino acids 266-291 and287-313 of Seq ID No 228; amino acids 85-114 of Seq ID No 229; aminoacids 36-64 and 83-109 of Seq ID No 230; amino acids 264-285 and 280-300of Seq ID No 231; amino acids 102-128 and 124-152 of Seq ID No 232;amino acids 404-429 and 445-468 of Seq ID No 233; amino acids 343-374and 370-401 of Seq ID No 234; amino acids 158-182 and 178-202 of Seq IDNo 235; amino acids 151-180 of Seq ID No 236; amino acids 549-579,575-605 and 601-630 of Seq ID No 237; amino acids-7-23 and 19-46 of SeqID No 238; amino acids 48-75 and 71-98 of Seq ID No 239; amino acids789-813 and 809-834 of Seq ID No 240; amino acids 759-783 of Seq ID No241; amino acids 160-188, 184-211 and 207-232 of Seq ID No 242; aminoacids 130-159 of Seq ID No 243; amino acids 117-147, 143-173 and 169-201of Seq ID No 244; amino acids 248-276, 272-300 and 296-323 of Seq ID No245; amino acids 21-43 of Seq ID No 246; amino acids 231-256 and 252-278of Seq ID No 247; amino acids 62-91, 87-115 and 199-227 of Seq ID No248; amino acids 116-141, 137-162 and 158-183 of Seq ID No 249; aminoacids 46-69, 65-87 and 82-105 of Seq ID No 250; amino acids 117-142 and138-163 of Seq ID No 251; amino acids 208-233 and 229-255 of Seq ID No252; amino acids 61-88 of Seq ID No 253; amino acids 99-124, 120-145 and141-167 of Seq ID No 254; amino acids 74-103, 99-128, 124-152 and148-176 of Seq ID No 255; amino acids 202-231 and 227-256 of Seq ID No256; amino acids 129-154 and 150-178 of Seq ID No 257; amino acids95-126 of Seq ID No 258; amino acids 226-256 and 252-282 of Seq ID No259; amino acids 171-198, 194-221 and 217-240 of Seq ID No 260; aminoacids 35-65 and 61-91 of Seq ID No 261; amino acids 608-631 of Seq ID No262; amino acids 124-149 of Seq ID No 263; amino acids-14-21 and 17-49of Seq ID No 264; amino acids 127-157 and 153-182 of Seq ID No 265;amino acids 150-176 of Seq ID No 266; amino acids 48-79 and 75-106 ofSeq ID No 267; amino acids 435-466 of Seq ID No 268; amino acids 151-180and 176-206 of Seq ID No 269; amino acids 126-151 and 167-190 of Seq IDNo 270; amino acids 89-118, 114-144 and 140-170 of Seq ID No 271; aminoacids 80-112 of Seq ID No 272; amino acids 9-36 of Seq ID No 274; aminoacids 117-140 of Seq ID No 276; amino acids 72-97, 93-117 and 113-137 ofSeq ID No 277; amino acids 723-746 of Seq ID No 278; amino acids 271-300of Seq ID No 279; amino acids 240-271 and 267-296 of Seq ID No 280;amino acids 165-188 and 183-206 of Seq ID No 281; amino acids 316-344and 340-364 of Seq ID No 282; amino acids-3-27 and 23-50 of Seq ID No283; amino acids 212-244, 532-561, 557-586 and 582-611 of Seq ID No 284;amino acids 276-302 and 298-324 of Seq ID No 285; amino acids 335-364and 360-389 of Seq ID No 286; amino acids 41-64 and 59-82 of Seq ID No287; amino acids 53-77 of Seq ID No 288; amino acids 13-37 of Seq ID No289; amino acids 69-94 and 90-114 of Seq ID No 290; amino acids 19-42and 37-60 of Seq ID No 291; amino acids 1-25 of Seq ID No 292; aminoacids 30-54 and 50-75 of Seq ID No 293; amino acids 111-135 of Seq ID No294; amino acids 25-54 of Seq ID No 295; amino acids 67-98 and 94-126 ofSeq ID No 334; amino acids 9-32, 27-51, 46-70 and 65-86 of Seq ID No335; amino acids 1-24 and 20-44 of Seq ID No 336; amino acids 58-82 ofSeq ID No 337; amino acids 37-62, 58-82 and 77-101 of Seq ID No 338;amino acids 37-68 and 64-93 of Seq ID No 339; amino acids 317-347 and343-375 of Seq ID No 340; amino acids 140-164 of Seq ID No 341; aminoacids 13-40, 36-60 and 55-79 of Seq ID No 342; amino acids 56-79 and75-101 of Seq ID No 343; amino acids 77-101 of Seq ID No 344; aminoacids 94-118 of Seq ID No 345; amino acids 46-105 of Seq ID No 205;amino acids 56-111 of Seq ID No 216; amino acids 25-107 of Seq ID No223; amino acids 19-66 of Seq ID No 224; amino acids 85-114 of Seq ID No229; amino acids 37-109 of Seq ID No 230; amino acids 266-296 of Seq IDNo 231; amino acids 103-152 of Seq ID No 232; amino acids 167-218 of SeqID No 235; amino acids 790-834 of Seq ID No 240; amino acids 761-781 ofSeq ID No 241; amino acids 176-232 of Seq ID No 242; amino acids 117-201of Seq ID No 244; amino acids 249-323 of Seq ID No 245; amino acids232-278 of Seq ID No 247; amino acids 209-255 of Seq ID No 252; aminoacids 75-176 of Seq ID No 255; amino acids 202-256 of Seq ID No 256;amino acids 130-178 of Seq ID No 257; amino acids 69-126 of Seq ID No258; amino acids 126-148 of Seq ID No 263; amino acids 1-49 of Seq ID No264; amino acids 127-182 of Seq ID No 265; amino acids 48-106 of Seq IDNo 267; amino acids 410-466 of Seq ID No 268; amino acids 152-206 of SeqID No 269; amino acids 555-621 of Seq ID No 275; amino acids 166-202 ofSeq ID No 281; amino acids 317-364 of Seq ID No 282; amino acids 1-50 ofSeq ID No 283; amino acids 277-324 of Seq ID No 285; amino acids 14-36of Seq ID No 289; amino acids 6-43 of Seq ID No 336; amino acids 57-101of Seq ID No 343; amino acids 84-95 of Seq ID No 344; and amino acids98-115 of Seq ID No
 345. 26. The antigen according to claim 25, wherebythe antigen further consists of a) 1 to 50 additional amino acidresidue(s); and/or b) at least one additional amino acid residueheterologous to the core amino acid sequence.
 27. (canceled)
 28. Theantigen according to claim 26, wherein the antigen comprises at least 2,at least 3, at least 4, at least 5 or at least 6 core amino acidsequences as defined in claim
 25. 29-38. (canceled)
 39. An antibody, orat least an effective part thereof, which binds to at least a selectivepart of an antigen or a fragment or variant thereof as defined in claim12. 40-59. (canceled)
 60. A method for the treatment of a Klebsiellainfection in an animal or human in need thereof, comprising the step ofadministering to said animal or human a therapeutically effective amountof an antigen, or an active fragment or an active variant thereof, asdefined in claim
 12. 61. A method for immunizing an animal or humanagainst infection with a Klebsiella organism, comprising the step ofadministering to said animal or human an effective amount of theantigen, or an active fragment or an active variant thereof, as definedclaim 12 or an antibody as defined in claim 39 wherein the effectiveamount is suitable to elicit an immune response in said animal or human.62-63. (canceled)